IJC International Journal of Cancer

Transcription

1 IJC International Journal of Cancer Vitamin B2 intake and colorectal cancer risk; results from the Nurses Health Study and the Health Professionals Follow-Up Study cohort Yeong Sook Yoon 1,2 *, Seungyoun Jung 3 *, Xuehong Zhang 4, Shuji Ogino 1,4,5, Edward L. Giovannucci 1,4 and Eunyoung Cho 4,6,7 1 Departments of Nutrition (YSY, ELG) and Departments of Epidemiology (SO, ELG), Harvard T.H. Chan School of Public Health, Boston, MA 2 Department of Family Medicine, Inje University Ilsan Paik Hospital, Goyang-Si, Gyeonggi-Do, Korea 3 Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD 4 Channing Division of Network Medicine, Department of Medicine (XZ, ELG, EC) and Department of Pathology (SO), Harvard Medical School, Brigham and Women s Hospital, Boston, MA 5 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 6 Department of Dermatology, Warren Alpert Medical School, Brown University, Providence, RI 7 Department of Epidemiology, Brown University School of Public Health, Providence, RI Vitamin B2 serves as a cofactor to enhance one-carbon metabolism, maintain mucous membranes, and has been implicated in lowering colorectal cancer (CRC) risk. However, few prospective studies have examined the association between vitamin B2 intake and CRC. In this study, we estimated the associations between vitamin B2 intake and CRC risk using the Nurses Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS) cohorts. Vitamin B2 intake was measured by a validated food frequency questionnaire every 4 years. Among 100,033 women in the NHS and 44,007 men in the HPFS we documented a total of 3,037 incident CRC cases (2,093 women and 944 men) during years of follow-up until Intakes of total (from food and supplements), dietary (from food only), and supplemental vitamin B2 were inversely related to CRC risk in age-adjusted analysis in NHS. However, the association was attenuated and no longer statistically significant in multivariate analysis (p-trend 0.08). The pooled multivariate relative risks (95% confidence interval) comparing individuals in the extreme quintiles of intakes were 0.93 ( ) for total vitamin B2, 0.89 ( ) for dietary vitamin B2 and 0.94 ( ) for supplemental vitamin B2. These associations of total vitamin B2 intake were similar for risk of CRC with varying lag-time periods (0 4, 4 8, 8 12 or years), for risk of CRC subtypes by tumor location, and across strata of intake of folate or alcohol. Our prospective data do not support a beneficial role of vitamin B2 intake in lowering incidence of CRC. Impairments in the one-carbon metabolism may affect methylation and synthesis of DNA, thereby influencing colorectal carcinogenesis. Within the one-carbon metabolism, vitamin B2 serves as a cofactor for methylenetetrahydrofolate reductase (MTHFR), a crucial enzyme enhancing DNA integrity by Key words: vitamin B2, colorectal neoplasms, cohort studies, alcohol, folate, diet, supplements, latency *Y.S.Y. and S.J. contributed equally to this work Grant sponsor: National Institutes of Health; Grant number: P01 CA87969, P01 CA55075, UM1 CA186107, UM1 CA167552, R01 CA151993, RO1 CA and R35 CA197735; Grant sponsor: Inje University ( ); Grant number: DOI: /ijc History: Received 5 Nov 2015; Accepted 1 Apr 2016; Online 15 Apr 2016 Correspondence to: Eunyoung Cho, ScD, Department of Dermatology, Warren Alpert Medical School, Brown University, 222 Richmond St, Providence, RI 02903, Tel.: , Fax: , eunyoung_cho@brown.edu mediating purine, and pyrimidine production, and is involved in epigenetic alteration by promoting the production of methyl donors required for DNA methylation. 1 Furthermore, vitamin B2 helps to maintain the mucosal membranes in the digestive system and upregulates the immune response by facilitating the conversion of tryptophan into niacin within the kynurenine metabolism. 1 Despite these mechanisms suggesting a potential role of B2 in colorectal carcinogenesis, epidemiological studies have been inconsistent. In contrast to null findings from most individual cohort studies 2 8 and case-control studies, 9 17 a recent metaanalyses found significant inverse association between vitamin B2 intake and risk of colorectal cancer (CRC) from four casecontrol studies [pooled odds ratio (OR) , 95% confidence interval (95% CI): for highest vs. lowest categories of intake of vitamins B2] and from five prospective cohort studies (pooled OR , 95% CI: ). 18,19 These suggest that limited statistical power might have been a reason for the null associations in individual studies. On the other hand, some of the studies included in the meta-analysis had a limited adjustment of risk factors of CRC and thus might have suffered

2 Yoon et al. 997 What s new? Some previous studies have indicated that vitamin B2 (riboflavin) may lower the risk of colorectal cancer (CRC). However, in this multivariate analysis of extensive data from the Nurses Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS), the authors did not find any significant benefit of vitamin B2 intake in lowering CRC risk, regardless of timing of intake, cancer subsite or other CRC-related nutrient intakes. residual confounding. Also, potential publication bias was noted among observational studies included in the metaanalyses. 18 Most previous studies have assessed vitamin B2 intake only from food sources and did not take into account intake from supplemental sources. 2 7,11,13,14,16,17 Only a few studies evaluated the associations between total vitamin B2 intake including supplements 9,10,12,15,20 or supplemental vitamin B2 intake itself 20 and CRC risk. In the Women s Health Initiative Observational study(whi-os) dietary vitamin B2 intake and supplemental vitamin B2 intake were not associated with risk of CRC, but total vitamin B2 intake was significantly associated with a lower risk of CRC (multivariate relative risk [RR] ; 95% CI , for >3.97 mg/d versus < 1.80 mg/d). 20 In addition, the effects of diet or nutrient intake as well as timing of exposure to these dietary factors (i.e., intake in the remote past or in the recent before diagnosis) might have different influences on CRC risk. For example, folate intake in the remote past was found to be most strongly inversely associated with risk of CRC, 21 which suggests the importance of timing of intake of one-carbon metabolism-related nutrients on the development of CRC. However, previous cohort studies 2 8,20 only had a single assessment of vitamin B2 intake. No study has examined the latency of vitamin B2 intake in relation to CRC risk. Furthermore, the association between vitamin B2 intake and CRC risk might be modified by other factors. Alcohol is known to disrupt the metabolism of vitamin B2 and induce DNA instability and abnormal DNA methylation. 22 Thus, alcohol intake might modify the association between vitamin B2 and CRC, but findings have been inconsistent. 2,6,9,20 One-carbon metabolism is a complex network that interconnects multiple nutrients. The association between vitamin B2 intake and CRC risk might be modified by folate 2,4,9,20 and other nutrients related to the onecarbon metabolism. Hence, we performed this study to evaluate the association of intakes of total (from foods and supplements), dietary (from foods only), and supplemental vitamin B2 with risk of CRC. Further, we evaluated whether the association varies according to the time between vitamin B2 intakes and CRC diagnosis and anatomic subsites and by several CRC risk factors including alcohol consumption, smoking status, multivitamin use, folate intake, calcium intake and family history of CRC. Methods Study design and participants The Nurses Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS) are ongoing prospective cohort studies. 21,23,24 The NHS was initiated in 1976 including 121,700 registered female nurses of aged 30 to 55 years. The HPFS was a prospective cohort study including 51,529 male health professionals aged years initiated it Participants responded to biennial follow-up questionnaires including demographic information, medical histories, lifestyles, and risk factors for chronic diseases. The follow-up of both cohorts was generally complete for more than 90%. For the current examination of vitamin B2 intake in relation to CRC risk, the baseline year was 1984 in the NHS and 1986 in HPFS, when vitamin B2 was first assessed from a comprehensive dietary food frequency questionnaire (FFQ) with 130 food items. We excluded participants who reported implausible calorie intake (<600 or >3,500 kcal/d for women and <800 or >4,200 kcal/d for men) or left more than 70 questions blank in the FFQ. We also excluded participants who were previously diagnosed with cancer (except non-melanoma skin cancer) and who had ulcerative colitis or Crohn s disease at baseline. Consequently, 100,033 women in the NHS and 44,007 men in the HPFS were included in this analysis and followed until These studies were approved by the institutional review board at the Harvard T.H. Chan School of Public Health and Brigham and Women s Hospital, Boston, MA. Assessment of dietary intake Dietary information was collected using a validated FFQ with 130 food items approximately every 2 4 years since 1984 in the NHS and since 1986 in the HPFS. 25 Nutrient intake was calculated by multiplying the frequency response of each specified food item by the nutrient content of the specified portion sizes and by summing these products for all food items. A food composition database from the US Department of Agriculture and other sources was used to estimate nutrient intake. 26,27 The use of multivitamins and other dietary supplements was ascertained via biennial questionnaires. Total vitamin B2 intake included intakes from foods and supplements, whereas dietary vitamin B2 intake included intakes from foods only. Intakes of vitamins B2 measured by FFQ have previously been validated. 25,28 30 For participants in NHS and HPFS, approximately 60% amount of total vitamin B2 intake was from supplements containing vitamin B2. Nutrient intakes were

3 998 Vitamin B2 intake and the risk of colorectal cancer cumulatively averaged and adjusted for energy intake using residual methods. 31 The correlation coefficients of total vitamin B2 intake and dairy foods (main food sources of vitamin B2) from FFQ and from two one-week diet records were 0.88 and 0.70 for men and 0.58 and 0.79 for women, 25,28 30 respectively. Ascertainment of CRC cases Newly diagnosed CRC cases from baseline until 2010 were identified through participants self-report on each biennial questionnaire, and confirmed through medical and pathological record review. The National Death Index was used to identify participants whose death was attributable to CRC. 32 Information on tumor location was extracted from medical records and pathology reports. A total of 2,093 incident CRC cases in the NHS and 944 cases in the HPFS were identified and included in the analysis. Assessment of confounding variables Information on potential confounders such as body mass index (BMI), physical activity, smoking status, aspirin use, hormone replacement therapy (only in NHS), multivitamin supplement use, family history of CRC, and history of sigmoidoscopy/colonoscopy were collected from self-reported biennial questionnaires. Statistical analysis We used cumulative average vitamin B2 intakes as our primary exposure 33 and also separately estimated baseline vitamin B2 intakes and intakes with a varying lag time before diagnosis of CRC to evaluate the latency between vitamin B2 intake and CRC. 21,23 For example, in the HPFS, for latency of 0 4 years before diagnosis, we used vitamin B2 intake in 1986 for cases diagnosed from 1986 through 1990; intake in 1990 for cases diagnosed from 1990 through 1994; and so forth. The lag time of vitamin B2 intake for non-cases was assessed in the same way relative to censor time. We categorized vitamin B2 intake into quintiles based on the intake distribution in our study population. A Cox proportional hazards model was used to estimate RR and 95% CI across quintiles of vitamin B2 intake for risk of overall CRC and anatomic subsites of CRC separately in NHS and HPFS. Person-years of follow-up for every analysis was calculated from the date of baseline questionnaire return (1984 for NHS; 1986 for HPFS) to the date of CRC diagnosis, death, loss to follow-up, or end of the follow-up (2010), whichever came first. Age at baseline (in months) and the calendar year of questionnaire return were included as stratification variables. The multivariate model adjusted for factors associated with CRC risk and vitamin B2 intakes as follows: BMI (<25, 25 <30 or 30, kg/m 2 ), pack-years of smoking before age 30 (continuous, pack-years), family history of colorectal cancer (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), physical activity (quintiles, MET-hrs/wk), aspirin use (yes or no), postmenopausal hormone use (premenopausal, never, past or current; NHS only), intake of total calorie (quintiles, Kcal/day), alcohol (<5, 5 <10, 10 <15, 15 <30 or 30, g/day), red meat (quintiles, serving/day), dietary calcium (quintiles, mg/day), dietary folate (quintiles, lg/day) and dietary vitamin D (quintiles, IU/day). Missing indicators were created for missing responses for each covariate, if applicable. Tests for linear trend were conducted using the median values of each quintile of vitamin B2 as continuous term and calculating Wald statistic of it. The study-specific RR was pooled using a random effects model. 34,35 The heterogeneity between two studies was assessed by calculating the Q statistic. 35 In sensitivity analyses, cases diagnosed within the first two years of follow-up were excluded to evaluate the potential influence of subclinical diseases. We also restricted our analyses to non-users of multivitamins or additionally adjusted for multivitamin use or other one-carbon nutrients such as methionine, vitamin B6, choline, vitamin B12 and betaine in multivariate models. We conducted analyses stratified by alcohol consumption, smoking status, multivitamin use, folate intake, calcium intake and family history of CRC. Test for effect modification was conducted by including the crossproduct term between a continuous term for vitamin B2 intake and categorical terms for stratification factors in the multivariate model. 36 All analyses were performed using the SAS software package (version 9.1). All tests were two-sided and p < 0.05 was considered statistically significant. Results In this prospective analysis including 3,037 CRC cases during years of follow-up from two cohorts, individuals with higher intake of total vitamin B2 were more likely to be physically active, use multivitamins and aspirins, and have greater intakes of dietary folate, calcium, and vitamin D (Table 1). The associations of total, dietary and supplemental vitamin B2 intakes with CRC are presented in Table 2. Women in the highest quintile of total, dietary, and supplemental vitamin B2 intake had an age-adjusted RR of 0.69(95% CI: , p-trend<0.001), 0.81(95% CI: , p-trend 50.01), and 0.70(95% CI: , p-trend <0.001) for CRC compared with women in the lowest quintile. However, these significant associations were attenuated and no longer statistically significant after further adjustments for life style and dietary factors; among those, calcium intake was the major confounder. The pooled multivariate RRs and 95% CI comparing individuals in the highest to the lowest quintiles of intake were 0.93 ( ) for total vitamin B2, 0.89( ) for dietary vitamin B2, and 0.94 ( ) for supplemental vitamin B2 (ptrend 0.59). Comparing extreme intakes of total vitamin B2 using deciles yielded similar results; the pooled multivariate RR was 0.82 ( ) comparing individuals in the highest to the lowest deciles (not shown in tables). Adjustment for both dietary and supplementary intakes of vitamin B2 simultaneously in the multivariate model did not change the results materially (data not shown). The association of dietary intake

4 Table 1. Age-standardized demographic, lifestyle, and dietary characteristics of participants in 1990 in the Nurses Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) according to quintiles of total vitamin B2 intake 1 Quintiles of total vitamin B2 intake Characteristics Q1 Q3 Q5 NHS Median intake (mg/day) No. of participants 17,692 17,865 17,509 Age (y) 55.6(7.1) 57.0(7.2) 57.4(7.1) BMI (kg/m 2 ) 25.7(5.0) 26.0(5.0) 25.5(4.9) Physical activity (MET-h/wk) (14.6) 14.2(16.1) 15.1(16.7) Pack years smoking before age (5.2) 3.5(5.2) 3.8(5.5) Endoscopy experience (%) Family history of colorectal cancer (%) Aspirin use (%) Current multivitamin use (%) Alcohol drinker (%) Dietary intake Total energy (Kcal/day) 1,621(441) 1,811(484) 1,703(465) Red meat intake (servings/day) 1.1(0.5) 1.1(0.5) 1.0(0.5) Fruit and vegetable intake (servings/day) 4.3(1.6) 5.3(2.0) 5.3(2.0) Alcohol consumption (g/day) 7.9(11.9) 6.1(9.0) 6.6(10.5) Vitamin D intake (IU/day) 178.3(100.6) 326.1(165.4) 460.9(308.5) Calcium intake (mg/day) 693.6(299) 1,015(328) 1,192(467) Folate intake (lg/day) 284.7(79.5) 408.1(157.2) 536.9(260.7) Whole milk (servings/day) 0.09(0.20) 0.18(0.44) 0.12(0.31) Skim milk (servings/day) 0.23(0.29) 1.07(0.86) 0.80(0.78) Breakfast cereal (servings/day) 0.15(0.20) 0.40(0.32) 0.32(0.30) HPFS Median intake (mg/day) No. of participants 8,642 8,647 8,572 Age (y) 56.1(9.1) 57.8(9.7) 58.7(9.6) BMI (kg/m 2 ) 25.7(3.0) 25.6(3.0) 25.4(3.0) Physical activity (MET-h/wk) 31.7(32.5) 36.1(36.7) 36.0(39.3) Pack years smoking before age (7.2) 4.7(7.0) 5.1(7.1) Endoscopy experience (%) Family history of colorectal cancer (%) Aspirin use (%) Current multivitamin use (%) Alcohol drinker (%) Dietary intake Total energy (Kcal/day) 1,777(457) 2,001(553) 1,858(492) Red meat intake (servings/day) 1.2(0.9) 1.3(0.9) 1.0(0.8) Fruit and vegetable intake (servings/day) 4.8(2.3) 5.8(2.7) 5.9(2.8) Alcohol consumption (g/day) 11.8(14.8) 10.1(11.9) 11.2(14.0) Vitamin D intake (IU/day) 256.5(123.7) 388.3(156.6) 574.6(358.8) Calcium intake (mg/day) 636(204) 967(302) 1,075(486) Folate intake (lg/day) 343.5(88.3) 445.8(145.2) 633.5(312.3) Whole milk (servings/day) 0.06(0.17) 0.19(0.53) 0.11(0.35) Skim milk (servings/day) 0.24(0.31) 1.17(0.99) 0.78(0.86) Breakfast cereal (servings/day) 0.19(0.26) 0.51(0.40) 0.41(0.40) Abbreviations: BMI: body mass index; MET-h: metabolic equivalent task hours. 1 Values are means (SD) or percentages, otherwise specified. All data except age are standardized to the age distribution of the study population. 2 MET-h 5 sum of the average time spent in each activity 3 the MET value of each activity.

5 1000 Vitamin B2 intake and the risk of colorectal cancer Table 2. Relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer according to quintiles of energy-adjusted vitamin B2 intake in the Nurses Health Study (NHS) and Health Professionals Follow-up Study (HPFS) 1 Q1 Q2 Q3 Q4 Q5 p-trend 2 p-between-studies the highest category 3 Total vitamin B2 Median intake, mg/day (NHS/HPFS) 1.6/ / / / /15.9 Person-years (NHS/HPFS) 434,225/185, ,112/191, ,128/192, ,581/189, ,051/189,712 No. of cases (NHS/HPFS) 455/ / / / /198 Age-adjusted RR (95% CI) NHS 1 (reference) 0.86 ( ) 0.87 ( ) 0.73 ( ) 0.69 ( ) <0.001 HPFS 1 (reference) 0.87 ( ) 0.77 ( ) 0.69 ( ) 0.84 ( ) 0.76 Pooled 1 (reference) 0.86 ( ) 0.84 ( ) 0.72 ( ) 0.75 ( ) Multivariable RR (95% CI) 4 NHS 1 (reference) 0.99 ( ) 1.11 ( ) 0.98 ( ) 0.93 ( ) 0.08 HPFS 1 (reference) 0.89 ( ) 0.80 ( ) 0.74 ( ) 0.91 ( ) 0.32 Pooled 1 (reference) 0.96 ( ) 0.95 ( ) 0.87 ( ) 0.93 ( ) Dietary vitamin B2 Median intake, mg/day (NHS/HPFS) 1.4/ / / / /2.7 Person-years (NHS/HPFS) 432,947/187, ,991/192, ,969/193, ,968/189, ,223/185,668 No. of cases (NHS/HPFS) 411/ / / / /167 Age-adjusted RR (95% CIs) NHS 1 (reference) 0.88 ( ) 0.93 ( ) 0.90 ( ) 0.81 ( ) 0.01 HPFS 1 (reference) 0.90 ( ) 0.78 ( ) 0.87 ( ) 0.67 ( ) <0.001 Pooled 1 (reference) 0.89 ( ) 0.87 ( ) 0.89 ( ) 0.75 ( ) Multivariable RR (95% CIs) NHS 1 (reference) 0.95 ( ) 1.07 ( ) 1.10 ( ) 1.06 ( ) 0.41 HPFS 1 (reference) 0.91 ( ) 0.81 ( ) 0.92 ( ) 0.73 ( ) 0.03 Pooled 1 (reference) 0.94 ( ) 0.95 ( ) 1.03 ( ) 0.89 ( ) Supplemental vitamin B2 Median intake mg/day (NHS/HPFS) 0/0 0.6/ / / /13.1 Person-years (NHS/HPFS) 781,167/369, ,550/101, ,176/198, ,317/188, ,888/90,380 No. of cases (NHS/HPFS) 736/ / / / /94 Age-adjusted RR (95% CIs) NHS 1 (reference) 0.97 ( ) 0.88 ( ) 0.80 ( ) 0.70 ( ) <0.001 HPFS 1 (reference) 0.95 ( ) 0.82 ( ) 0.84 ( ) 0.96 ( ) 0.69

6 Yoon et al Table 2. Relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer according to quintiles of energy-adjusted vitamin B2 intake in the Nurses Health Study (NHS) and Health Professionals Follow-up Study (HPFS) (Continued) p-between-studies the highest category 3 Q1 Q2 Q3 Q4 Q5 p-trend 2 Pooled 1 (reference) 0.96 ( ) 0.86 ( ) 0.81 ( ) 0.81 ( ) Multivariable RR (95% CIs) NHS 1 (reference) 1.09 ( ) 1.08 ( ) 1.01 ( ) 0.91 ( ) 0.09 HPFS 1 (reference) 0.93 ( ) 0.84 ( ) 0.88 ( ) 1.01 ( ) 0.49 Pooled 1 (reference) 1.04 ( ) 0.96 ( ) 0.96 ( ) 0.94 ( ) A Cox proportional hazards model was used and values are relative risk and 95% confidence intervals in parenthesis. 2 Two sided; calculated by using Wald s test statistic. 3 Two sided; calculated by using the Q test statistic. 4 Adjusted for BMI (<25, 25 <30, or 30, kg/m 2 ), pack-years of smoking before age 30 (continuous, pack-years), family history of colorectal cancer (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), physical activity (quintiles, MET-hrs/wk), aspirin use (yes or no), postmenopausal hormone use (premenopausal, never, past, or current; NHS only), intake of total calorie (quintiles, Kcal/day), alcohol (<5, 5 <10, 10 <15, 15 <30, or 30, g/day), red meat (quintiles, serving/day), dietary calcium (quintiles, mg/day), dietary folate (quintiles, lg/day) and dietary vitamin D (quintiles, IU/day); age in months and year of questionnaire return were included as stratification variables. of vitamin B2 with CRC risk significantly differed between men and women (p-heterogeniety50.01) and men in the highest quintile of dietary vitamin B2 intake, compared to those in the lowest quintile, had a multivariate RR of 0.73(95% CI: , p-trend 50.03), whereas no significant association was observed in women. When we evaluated the varying latent time period for the association between total vitamin B2 intake and CRC risk (Table 3), total vitamin B2 intake was not significantly associated with risk of CRC regardless of the length of lag-time between vitamin B2 intake and CRC diagnosis, although there was some suggestion of inverse association with longer time lag. The pooled multivariate RRs and 95% CI comparing individuals in the highest versus lowest quintiles of total vitamin B2 intake were 1.01 ( ) for intake 0 4 years before diagnosis, 0.95 ( ) for intake 4 8 years before diagnosis, 0.92 ( ) for intake 8 12 years before diagnosis and 0.88 ( ) for intake years before diagnosis (all P-trend0.65). The corresponding RR (95% CI) was 0.87 ( ) for baseline intake. Total vitamin B2 intake was not significantly associated with any specific anatomic subsite of CRC (Table 4). Intakes of alcohol, folate or calcium, multivitamin use, smoking status, calcium intake or family history of CRC also did not significantly modify the association observed between total vitamin B2 intake and risk of CRC (p-interaction 0.07) (Table 5). In our sensitivity analyses, restricting analyses to nonusers of multivitamins or excluding cases diagnosed within the first two years of follow-up did not change our main results (data not shown). Additional adjustment for intakes of fruits and vegetables, race, multivitamin use (yes or no), or other one-carbon nutrients such as methionine, vitamin B6, choline, vitamin B12 and betaine in multivariate models also yielded similar results (data not shown). Discussion We found that total vitamin B2 intake was not associated with overall CRC risk. This association was not modified by timing of intake and cancer subsites, although there was some suggestion of inverse association with longer time lag. There was no significant interaction with alcohol consumption, folate intake, calcium intake, smoking status, family history of CRC or multivitamin use for CRC risk. In this study, dietary, supplemental, and total vitamin B2 intakes were not significantly associated with overall risk of CRC. Our largely null results using cumulative updated vitamin B2 intake are not accordance with the findings of a recent meta-analysis which found a significantly lower CRC risk with high vitamin B2 intake (pooled OR [95% CI: ]). 18 However, our results were statistically significant in age-adjusted analysis, and these significant results were attenuated in multivariate analysis after adjusting for multiple dietary factors including intakes of alcohol, calcium, folate and red meat. The studies included in the meta-

7 1002 Vitamin B2 intake and the risk of colorectal cancer Table 3. Multivariate adjusted relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer using various lag time according to quintiles of energy-adjusted total vitamin B2 intake in the Nurses Health Study (NHS) and Health Professionals Follow-up Study (HPFS) 1 Quintiles of total vitamin B2 intake p-between-studies Q1 Q2 Q3 Q4 Q5 p-trend 2 the highest category 3 Baseline Person-years (NHS/HPFS) 333,103/189, ,427/190, ,055/186, ,484/183, ,491/182,283 No. of cases (NHS/HPFS) 317/ / / / /179 NHS 1 (reference) 1.01 ( ) 1.01 ( ) 1.00 ( ) 0.89 ( ) 0.08 HPFS 1 (reference) 0.93 ( ) 0.78 ( ) 0.79 ( ) 0.84 ( ) 0.56 Pooled 1 (reference) 0.98 (0.86, 1.11) 0.90 ( ) 0.90 ( ) 0.87 ( ) Simple update (0 4-y lag) Person-years (NHS/HPFS) 424,479/183, ,115/188, ,795/191, ,822/195, ,887/188,627 No. of cases (NHS/HPFS) 469/ / / / /188 NHS 1 (reference) 0.89 ( ) 1.03 ( ) 0.98 ( ) 0.99 ( ) 0.84 HPFS 1 (reference) 1.03 ( ) 1.01 ( ) 0.98 ( ) 1.06 ( ) 0.39 Pooled 1 (reference) 0.93 ( ) 1.02 ( ) 0.98 ( ) 1.01 ( ) y lag Person-years (NHS/HPFS) 346,632/151, ,332/153, ,437/153, ,213/154, ,940/151,055 No. of cases (NHS/HPFS) 377/ / / / /144 NHS 1 (reference) 1.06 ( ) 1.07 ( ) 1.07 ( ) 0.96 ( ) 0.23 HPFS 1 (reference) 1.01 ( ) 0.91 ( ) 0.92 ( ) 0.93 ( ) 0.70 Pooled 1 (reference) 1.04 ( ) 1.02 ( ) 1.02 ( ) 0.95 ( ) y lag Person-years (NHS/HPFS) 272,030/121, ,259/121, ,731/118, ,832/117, ,057/116,363 No. of cases (NHS/HPFS) 322/ / / / /121 NHS 1 (reference) 1.09 ( ) 0.98 ( ) 0.98 ( ) 0.91 ( ) 0.12 HPFS 1 (reference) 0.98 ( ) 0.96 ( ) 0.89 ( ) 0.95 ( ) 0.98 Pooled 1 (reference) 1.06 ( ) 0.97 ( ) 0.96 ( ) 0.92 ( ) y lag Person-years (NHS/HPFS) 201,928/89,533 20,6135/89, ,893/86, ,598/85, ,219/83,508 No. of cases (NHS/HPFS) 251/ / /75 193/65 182/81 NHS 1 (reference) 1.13 ( ) 1.05 ( ) 0.92 ( ) 0.89 ( ) 0.27

8 Yoon et al Table 3. Multivariate adjusted relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer using various lag time according to quintiles of energy-adjusted total vitamin B2 intake in the Nurses Health Study (NHS) and Health Professionals Follow-up Study (HPFS) (Continued) Quintiles of total vitamin B2 intake p-between-studies Q1 Q2 Q3 Q4 Q5 p-trend 2 the highest category 3 HPFS 1 (reference) 0.92 ( ) 0.77 ( ) 0.72 ( ) 0.88 ( ) 0.62 Pooled 1 (reference) 1.05 ( ) 0.93 ( ) 0.86( ) 0.88 ( ) A Cox proportional hazards model was used and values are relative risk and 95% confidence intervals in parenthesis. Adjusted for BMI (<25, 25 <30, or 30, kg/m 2 ), pack-years of smoking before age 30 (continuous, pack-years), family history of colorectal cancer (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), physical activity (quintiles, MET-hrs/wk), aspirin use (yes or no), postmenopausal hormone use (premenopausal, never, past, or current; NHS only), intake of total calorie (quintiles, Kcal/day), alcohol (<5, 5 <10, 10 <15, 15 <30, or 30, g/day), red meat (quintiles, serving/day), dietary calcium (quintiles, mg/day), dietary folate (quintiles, lg/day) and dietary vitamin D (quintiles, IU/day); age in months and year of questionnaire return were included as stratification variables. 2 Two sided; calculated by using Wald s test statistic. 3 Two sided; calculated by using the Q test statistic. analysis had a limited adjustment of these dietary factors and thus might have suffered some residual confounding due to these factors. Nonetheless, the inverse association reported in the meta-analysis was similar to our result using baseline vitamin B2 intake (pooled RR [95% CI: ]). For participants in NHS and HPFS, median total vitamin B2 intakes were 3.2 mg/d for men and 3.0 mg/d for women. Considering that recommended dietary allowance (RDA) of vitamin B2 are 1.3 mg/d for men and 1.1 mg/d for women, our participants vitamin B2 intakes were high; even individuals in our lowest quintile consumed in excess of the RDA. Although there is a possibility of measurement error in diet assessment, this information suggests that our cohort consist of a well-nourished study population. Furthermore, the ranges of vitamin B2 intake and median values of vitamin B2 intake in lowest quantiles in the studies included in the meta-analysis except for Shanghai Women s Health Study, 4 in which vitamin B2 intake was relatively lower than those in our study, were comparable to ours. 18 Therefore, the differences in the level of intake could not explain the differences of the results between the meta-analysis and ours. Among sources of vitamin B2 intake, dairy products (e.g., whole milk, skim milk and yogurt) accounted for 8.1% of intake in NHS and 7.6% in HPFS. Breakfast cereals accounted for 3.4% for NHS and 3.8% for HPFS. Our non-significant associations of vitamin B2 with CRC risk might be due to the fact that most of our study participants had already achieved potentially protective levels of vitamin B2; nonetheless, risk estimates were relatively flat even when comparing individual in higher deciles to those in the lowest decile of vitamin B2 intake. Other nutrient profiles within the main food sources of vitamin B2 might also have contributed to our result. Future large studies including populations with lower vitamin B2 intake or vitamin B2 status are warranted. Alternatively, since excess vitamin B2 is excreted primarily in the urine and very little is stored in the body, higher intakes (more than RDA) may not reflect the actual bioavailable vitamin B2. Few studies have evaluated possible associations between the risk of CRC and plasma levels of riboflavin or flavin mononucleotide (FMN) 37,38 and results were inconsistent. The European Prospective Investigation into Cancer and Nutrition (EPIC) study indicated that higher plasma levels of vitamin B2 were inversely associated with CRC (highest to lowest quintiles RR50.71[95% CI: ], p- trend50.02). 37 However, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort reported that serum riboflavin was unrelated to CRC risk. 38 Future evaluations of a possible association between circulating levels of vitamin B2 and risk of CRC are needed. To our knowledge, our study is the first prospective cohort study to examine vitamin B2 intake with varying lagtime in relation to CRC risk. We observed no significant association between intake of total vitamin B2 and risk of CRC by the lag-time between intake and diagnosis, although there was some suggestion of inverse association with longer

9 1004 Vitamin B2 intake and the risk of colorectal cancer Table 4. Multivariate adjusted relative risk (RR) and 95% confidence intervals (CIs) of anatomic subsite of colorectal cancer according to quintiles of energy-adjusted total vitamin B2 intake in the Nurses Health Study (NHS) and Health Professionals Follow-up Study (HPFS) 1 Quintiles of total vitamin B2 intake p-between-studies Q1 Q2 Q3 Q4 Q5 p-trend 2 the highest category 3 Proximal colon cancer No. of cases (NHS/HPFS) 193/52 173/71 201/72 174/64 157/60 NHS 1 (reference) 0.87 ( ) 1.05 ( ) 0.93 ( ) 0.87 ( ) 0.20 HPFS 1 (reference) 1.11 ( ) 1.03 ( ) 0.87 ( ) 0.90 ( ) 0.44 Pooled 1 (reference) 0.93 ( ) 1.04 ( ) 0.92 ( ) 0.87 ( ) Distal colon cancer No. of cases (NHS/HPFS) 98/66 106/63 122/44 85/53 95/68 NHS 1 (reference) 1.18 ( ) 1.47 ( ) 1.04 ( ) 1.24 ( ) 0.78 HPFS 1 (reference) 1.02 ( ) 0.72 ( ) 0.82 ( ) 1.14 ( ) 0.09 Pooled 1 (reference) 1.12 ( ) 1.05 ( ) 0.95 ( ) 1.20 ( ) Rectal cancer No. of cases (NHS/HPFS) 98/43 81/36 79/45 88/32 64/44 NHS 1 (reference) 0.93 ( ) 0.94 ( ) 1.07 ( ) 0.80 ( ) 0.15 HPFS 1 (reference) 0.67 ( ) 0.76 ( ) 0.57 ( ) 0.82 ( ) 0.41 Pooled 1 (reference) 0.83 ( ) 0.88 ( ) 0.81 ( ) 0.81 ( ) A Cox proportional hazards model was used and values are relative risk and 95% confidence intervals in parenthesis. Adjusted for BMI (<25, 25 <30, or 30, kg/m 2 ), pack-years of smoking before age 30 (continuous, pack-years), family history of colorectal cancer (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), physical activity (quintiles, MET-hrs/wk), aspirin use (yes or no), postmenopausal hormone use (premenopausal, never, past or current; NHS only), intake of total calorie (quintiles, Kcal/day), alcohol (<5, 5 <10, 10 <15, 15 <30, or 30, g/day), red meat (quintiles, serving/day), dietary calcium (quintiles, mg/day), dietary folate (quintiles, lg/day) and dietary vitamin D (quintiles, IU/day); age in months and year of questionnaire return were included as stratification variables. 2 Two sided; calculated by using Wald s test statistic. 3 Two sided; calculated by using the Q test statistic.

10 Yoon et al Table 5. Pooled multivariate relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer according to quintiles of energy-adjusted total vitamin B2 intake stratified by alcohol consumption, folate intake, multivitamin use, smoking status, or calcium intake 1 Quintiles of total vitamin B2 intake p-between-studies Q1 Q2 Q3 Q4 Q5 p-trend 2 the highest category 3 p-interaction 4 Alcohol consumption Nondrinkers No. of cases (NHS/HPFS) 216/41 175/41 208/37 161/44 150/57 RR (95% CIs) 1 (reference) 0.80 (0.65, 0.98) 0.76 ( ) 0.80 ( ) 0.82 ( ) > 0 15 g/day No. of cases (NHS/HPFS) 185/84 206/ /92 174/76 154/72 RR (95% CI) 1 (reference) 1.09 ( ) 1.06 ( ) 0.90 ( ) 0.87 ( ) g/day No. of cases (NHS/HPFS) 54/72 48/46 48/56 58/51 53/69 RR (95% CIs) 1 (reference) 0.98 ( ) 1.13 ( ) 1.22 ( ) 1.33 ( ) Folate intake < 400 lg/day No. of cases (NHS/HPFS) 283/99 206/69 145/49 91/17 87/22 RR (95% CIs) 1 (reference) 0.89 ( ) 1.01 ( ) 1.04 ( ) 0.95 ( ) lg/day No. of cases (NHS/HPFS) 172/98 223/ / / /176 RR (95% CIs) 1 (reference) 0.94 ( ) 0.91 ( ) 0.82 ( ) 0.86 ( ) Multivitamin use Ever No. of cases (NHS/HPFS) 206/ / / /196 RR (95% CIs) 1 (reference) 0.88 ( ) 0.88 ( ) 0.82 ( ) 0.88 ( ) Never No. of cases (NHS/HPFS) 249/ /87 90/47 11/11 8/2 RR (95% CIs) 1 (reference) 1.02 ( ) 1.18 ( ) 1.05 ( ) 0.75 ( ) Smoking status Ever No. of cases (NHS/HPFS) 280/ / / / /122 RR (95% CIs) 1 (reference) 0.97 ( ) 1.08 ( ) 0.95 ( ) 1.03 ( ) Never No. of cases (NHS/HPFS) 175/68 168/88 184/68 146/66 141/69 RR (95% CIs) 1 (reference) 0.92 ( ) 0.86 ( ) 0.80 ( ) 0.80 ( )

11 1006 Vitamin B2 intake and the risk of colorectal cancer Table 5. Pooled multivariate relative risk (RR) and 95% confidence intervals (CIs) of colorectal cancer according to quintiles of energy-adjusted total vitamin B2 intake stratified by alcohol consumption, folate intake, multivitamin use, smoking status, or calcium intake (Continued) Quintiles of total vitamin B2 intake p-between-studies Q1 Q2 Q3 Q4 Q5 p-trend 2 the highest category 3 p-interaction 4 Calcium intake < 1,000 mg/day No. of cases (NHS/HPFS) 383/ / / /88 134/94 RR (95% CIs) 1 (reference) 0.94 ( ) 0.93 ( ) 0.86 ( ) 0.99 ( ) ,000 mg/day No. of cases (NHS/HPFS) 72/6 146/34 249/82 243/83 223/104 RR (95% CIs) 1 (reference) 1.04 ( ) 1.12 ( ) 0.99 ( ) 0.90 ( ) Family history of colorectal cancer Yes No. of cases (NHS/HPFS) 91/35 80/39 94/33 81/33 82/33 RR (95% CIs) 1 (reference) 0.96 ( ) 1.08 ( ) 0.98 ( ) 1.07 ( ) No No. of cases (NHS/HPFS) 364/ / / / /165 RR (95% CIs) 1 (reference) 0.94 ( ) 0.95 ( ) 0.86 ( ) 0.92 ( ) A Cox proportional hazards model was used and values are relative risk and 95% confidence intervals in parenthesis. Adjusted for BMI (<25, 25 <30, or 30, kg/m 2 ), pack years of smoking before age 30 (continuous, pack-years), family history of colorectal cancer (yes or no), history of sigmoidoscopy/colonoscopy (yes or no), physical activity (quintiles, MET-hrs/wk), aspirin use (yes or no), postmenopausal hormone use (premenopausal, never, past or current; NHS only), intake of total calorie (quintiles, Kcal/day), alcohol (<5, 5 <10, 10 <15, 15 <30, or 30, g/day), red meat (quintiles, serving/day), dietary calcium (quintiles, mg/day), dietary folate (quintiles, lg/day) and dietary vitamin D (quintiles, IU/day); age in months and year of questionnaire return were included as stratification variables. 2 Two sided; calculated by using Wald s test statistic. 3 Two sided; calculated by using the Q test statistic. 4 p-interaction values were calculated by using Wald s test.

12 Yoon et al lag time. Most previous cohort studies 2,4 7,20 examined the associations between baseline intakes of vitamin B2 and CRC risk with varying years of follow-up (5.74 to 19 years) showed no associations, 2,4 7 though not all. 20 Among those studies, the Melbourne Collaborative Cohort Study 2 had the longest years of follow-up (19 years). This study also reported that baseline dietary intake of vitamin B2 was not associated with risk of CRC. 2 In our cohorts, folate intake in the remote past was more strongly inversely associated with risk of CRC, 21 but intake of vitamin B6 (which acts as a cofactor in the one-carbon metabolism) was not associated with CRC regardless of latency. 23 The co-factors in the one-carbon metabolism such as vitamin B2 and vitamin B6 might be weakly associated with risk of CRC compared to folate. Although the results were not statistically significant, our study showed lower risk of CRC associated with greater intake of baseline total vitamin B2. Vitamin B2 intake might be important in the initiation of colorectal carcinogenesis. 39 Findings regarding the effects of timing and duration of vitamin intake on CRC are inconclusive and warrant more research. The results of studies examining possible interactions of alcohol intake with vitamin B2 for risk of CRC are inconsistent. The WHI-OS, 20 a prospective cohort study among postmenopausal women, found that higher total vitamin B2 intake was significantly associated with 43% lower risk of CRC among current drinkers who consumed <13 g of alcohol per week. This partly disagrees with the results from the EPIC study, which showed a stronger inverse association for CRC with serum vitamin B2 among individuals with alcohol consumption 30 g/d. 37 However, other two cohort 2,6 and two case-control studies 9,17 showed no interaction between vitamin B2 and alcohol intake. We also did not observe a significant interaction by alcohol consumption (p-interaction50.07). It is not clear whether vitamin B2 intake mitigates disturbances in the one-carbon metabolism by alcohol intake; furthermore, the threshold of alcohol intake that can be nullified by vitamin B2 remains unclear. Vitamin B2 is closely related to folate and the effect of vitamin B2 in modulating the bioavailability of methyl groups might vary according to the folate status and genotype of MTHFR. 40 We found that an association between vitamin B2 and risk of CRC was not significantly modified by folate intake. This is consistent with previous results from one case-control 9 and three cohort studies. 2,4,20 On the other hand, the EPIC study 37 showed that the association between plasma vitamin B2 and CRC was modified by folate status (p-interaction ). Of interest are two case-control studies that suggested a protective effect of an interaction between vitamin B2 and the TT variant of the MTHFR C677T polymorphism to reduce the risk of CRC. 12,41 In light of results suggesting a modulating effect of MTHFR C677T polymorphism and considerable ethnic variations in the frequency of the MTHFR C677T genotype, 42 a large collaborative cohort study is warranted to evaluate an association between vitamin B2 intake and risk of CRC by genetic polymorphism of MTHFR. We did not observe site-specific differences in the association of vitamin B2 intake with CRC; however, several previous studies 2,6,37 showed that the association of vitamin B2 with CRC was stronger for colon cancer than for rectal cancer. Nevertheless, it is unclear whether vitamin B2 is more clearly associated with CRC in specific anatomical subsites as well as whether lower intake of vitamin B2 might render the colonic mucosa more sensitive to impairment of DNA methylation. This study has several strengths. To our knowledge, our study is the largest prospective cohort study that examined vitamin B2 intake and CRC. The repeated assessment of diet during follow-up allowed us to estimate the long-term intake of vitamin B2 during follow-up as well as intakes with various lag-time periods before diagnosis of CRC. In addition, with detailed information on diet and lifestyle factors, we were able to finely adjust for potential confounding factors for CRC. There are also limitations to our study. First, given the relatively high intakes of vitamin B2 as well as the high proportion of users of multivitamins containing vitamin B2 in our cohorts (39.4% for men and 38.1% for women), our study population was already well-nourished with the vitamin, which could have masked the effect of dietary B2 intake on CRC risk. However, the distribution of total vitamin B2 intake in our study was similar to that in a study of nationally representative sample of US population. 43 Examining associations among non-users of multivitamins and adjusting for multivitamin use also did not change our results. Second, we could not examine the effects of vitamin B2 intake in early life (i.e., childhood, adolescence or early adulthood), which might be important in colorectal carcinogenesis. Third, we had limited power for interaction analyses. Fourth, although majority of our study population were White, it is currently unknown if association between vitamin B2 intake and risk of CRC varies by ethnicity. Fifth, we cannot rule out residual confounding, although additionally adjusting for other one-carbon nutrients such as methionine, vitamin B6, choline, vitamin B12 or betaine in multivariate models, did not change our results. In conclusion, this prospective study indicates that intake of total vitamin B2 is not associated with overall CRC risk. The association between vitamin B2 intake and CRC risk was not modified by timing of intake during adulthood, cancer subsites or other CRC-related nutrient intakes. Further studies that evaluate the role of plasma levels of vitamin B2 and early-life vitamin B2 intake and their interaction with genetic variants within one-carbon metabolism on CRC risk are warranted. Acknowledgements Author contributions were as follows; E.C. and S.J. planned study design; S.J. and S.O. collected data; S.J. and X.Z. performed statistical analysis. Y.S.Y. wrote manuscript. E.C., S.O., E.L.G., X.Z. and S.J. provided critical revision of the manuscript. All authors contributed to the data interpretation, editing,

13 1008 Vitamin B2 intake and the risk of colorectal cancer and reviewing and approved the final manuscript. The funders had no role in design or conduct of the study; the collection, analysis, or interpretation of the data; the preparation, review, or approval of the manuscript. We would like to thank the participants and staff of the NHS and the HPFS for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and interpretation of these data. Disclosure The authors declare no conflicts of interest. REFERENCE 1. Powers HJ. Riboflavin (vitamin B-2) and health. Am J Clin Nutr 2003;77: Bassett JK, Severi G, Hodge AM, et al. Dietary intake of B vitamins and methionine and colorectal cancer risk. Nutr Cancer 2013;65: Key TJ, Appleby PN, Masset G, et al. Vitamins, minerals, essential fatty acids and colorectal cancer risk in the United Kingdom Dietary Cohort Consortium. Int J Cancer 2012;131:E Shrubsole MJ, Yang G, Gao YT, et al. Dietary B vitamin and methionine intakes and plasma folate are not associated with colorectal cancer risk in Chinese women. Cancer Epidemiol Biomarkers Prev 2009;18: Kabat GC, Miller AB, Jain M, et al. Dietary intake of selected B vitamins in relation to risk of major cancers in women. Br J Cancer 2008;99: de Vogel S, Dindore V, van Engeland M, et al. Dietary folate, methionine, riboflavin, and vitamin B-6 and risk of sporadic colorectal cancer. J Nutr 2008;138: Shin A, Li H, Shu XO, et al. Dietary intake of calcium, fiber and other micronutrients in relation to colorectal cancer risk: results from the Shanghai Women s Health Study. Int J Cancer 2006;119: de Vogel S, Bongaerts BW, Wouters KA, et al. Associations of dietary methyl donor intake with MLH1 promoter hypermethylation and related molecular phenotypes in sporadic colorectal cancer. Carcinogenesis 2008;29: Sharp L, Little J, Brockton NT, et al. Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, intakes of folate and related B vitamins and colorectal cancer: a case-control study in a population with relatively low folate intake. Br J Nutr 2008;99: Murtaugh MA, Curtin K, Sweeney C, et al. Dietary intake of folate and co-factors in folate metabolism, MTHFR polymorphisms, and reduced rectal cancer. Cancer Causes Control 2007;18: Otani T, Iwasaki M, Hanaoka T, et al. Folate, vitamin B6, vitamin B12, and vitamin B2 intake, genetic polymorphisms of related enzymes, and risk of colorectal cancer in a hospital-based case-control study in Japan. Nutr Cancer 2005;53: Le Marchand L, Donlon T, Hankin JH, et al. B- vitamin intake, metabolic genes, and colorectal cancer risk (United States). Cancer Causes Control 2002;13: La Vecchia C, Braga C, Negri E, et al. Intake of selected micronutrients and risk of colorectal cancer. Int J Cancer 1997;73: van Lee L, Heyworth J, McNaughton S, et al. Selected dietary micronutrients and the risk of right- and left-sided colorectal cancers: a casecontrol study in Western Australia. Ann Epidemiol 2011;21: Sun Z, Zhu Y, Wang PP, et al. Reported intake of selected micronutrients and risk of colorectal cancer: results from a large population-based casecontrol study in Newfoundland, Labrador and Ontario, Canada. Anticancer Res 2012;32: Morita M, Yin G, Yoshimitsu S, et al. Folaterelated nutrients, genetic polymorphisms, and colorectal cancer risk: the fukuoka colorectal cancer study. Asian Pac J Cancer Prev 2013;14: Jedrychowski W, Steindorf K, Popiela T, et al. Alcohol consumption and the risk of colorectal cancer at low levels of micronutrient intake. Med Sci Monit 2002;8:CR Liu Y, Yu QY, Zhu ZL, et al. Vitamin b2 intake and the risk of colorectal cancer: a meta- analysis of observational studies. Asian Pac J Cancer Prev 2015;16: Liu Y, Yu Q, Zhu Z, et al. Vitamin and multiplevitamin supplement intake and incidence of colorectal cancer: a meta-analysis of cohort studies. Med Oncol 2015;32: Zschabitz S, Cheng TY, Neuhouser ML, et al. B vitamin intakes and incidence of colorectal cancer: results from the Women s Health Initiative Observational Study cohort. Am J Clin Nutr 2013;97: Lee JE, Willett WC, Fuchs CS, et al. Folate intake and risk of colorectal cancer and adenoma: modification by time. Am J Clin Nutr 2011;93: Kruman II, Fowler AK. Impaired one carbon metabolism and DNA methylation in alcohol toxicity. J Neurochem 2014;129: Zhang X, Lee JE, Ma J, et al. Prospective cohort studies of vitamin B-6 intake and colorectal cancer incidence: modification by time? Am J Clin Nutr 2012;96: Colditz GA, Hankinson SE. The Nurses Health Study: lifestyle and health among women. Nat Rev Cancer 2005;5: Rimm EB, Giovannucci EL, Stampfer MJ, et al. Reproducibility and validity of an expanded selfadministered semiquantitative food frequency questionnaire among male health professionals. Am J Epidemiol 1992;135: discussion US Department of Agriculture Composition of foods raw, processed, and prepared Agricultural Handbook No. 8 Series. Washington, DC: Department of Agriculture, Government Printing Office, US Department of Agriculture USDA database for the choline content of common foods. Washington, DC: US Department of Agriculture, Feskanich D, Rimm EB, Giovannucci EL, et al. Reproducibility and validity of food intake measurements from a semiquantitative food frequency questionnaire. J Am Diet Assoc 1993;93: Willett WC, Sampson L, Stampfer MJ, et al. Reproducibility and validity of a semiquantitative food frequency questionnaire. Am J Epidemiol 1985;122: Jung S, Je Y, Giovannucci EL, et al. Derivation and validation of homocysteine score in u.s. Men and women. J Nutr 2015;145: Willett W, Stampfer MJ. Total energy intake: implications for epidemiologic analyses. American J Epidemiol 1986;124: Stampfer MJ, Willett WC, Speizer FE, et al. Test of the National Death Index. Am J Epidemiol 1984;119: Hu FB, Stampfer MJ, Rimm E, et al. Dietary fat and coronary heart disease: a comparison of approaches for adjusting for total energy intake and modeling repeated dietary measurements. Am J Epidemiol 1999;149: Laird NM, Ware JH. Random-effects models for longitudinal data. Biometrics 1982;38: DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7: Hosmer DW. Applied logistic regression, 3rd edn. Hosmer DW, Lemeshow S, Sturdivant RX, eds. Wiley, Eussen SJ, Vollset SE, Hustad S, et al. Plasma vitamins B2, B6, and B12, and related genetic variants as predictors of colorectal cancer risk. Cancer Epidemiol Biomarkers Prev 2010;19: Weinstein SJ, Albanes D, Selhub J, et al. One-carbon metabolism biomarkers and risk of colon and rectal cancers. Cancer Epidemiol Biomarkers Prev 2008;17: van der Pols JC, Bain C, Gunnell D, et al. Childhood dairy intake and adult cancer risk: 65-y follow-up of the Boyd Orr cohort. Am J Clin Nutr 2007;86: Powers HJ. Interaction among folate, riboflavin, genotype, and cancer, with reference to colorectal and cervical cancer. J Nutr 2005;135:2960S 6S. 41. van den Donk M, Buijsse B, van den Berg SW, et al. Dietary intake of folate and riboflavin, MTHFR C677T genotype, and colorectal adenoma risk: a Dutch case-control study. Cancer Epidemiol Biomarkers Prev 2005;14: Brockton NT. Localized depletion: the key to colorectal cancer risk mediated by MTHFR genotype and folate? Cancer Causes Control 2006;17: Fulgoni VL, III, Keast DR, Bailey RL, et al. Foods, fortificants, and supplements: Where do Americans get their nutrients? J Nutr 2011; 141: