Folate, vitamin B 6, and vitamin B 12 are cofactors in

Research Letters Dietary Folate and Vitamin B 6 and B 12 Intake in Relation to Mortality From Cardiovascular Diseases Japan Collaborative Cohort Study Renzhe Cui, MD; Hiroyasu Iso, MD; Chigusa Date, MD; Shogo Kikuchi, MD; Akiko Tamakoshi, MD; for the Japan Collaborative Cohort Study Group Background and Purpose The association of dietary folate and B vitamin intakes with risk of cardiovascular disease is controversial, and the evidence in Asian populations is limited. Methods A total of 23 119 men and 35 611 women, age 40 to 79 years, completed a food frequency questionnaire in the Japan Collaborative Cohort Study. During the median 14-year follow-up, there were 986 deaths from stroke, 424 from coronary heart disease, and 2087 from cardiovascular disease. Results Dietary folate and vitamin B 6 intakes were inversely associated with mortality from heart failure for men and with mortality from stroke, coronary heart disease, and total cardiovascular disease for women. These inverse associations did not change materially after adjustment for cardiovascular risk factors. No association was found between vitamin B 12 intake and mortality risk. Conclusions High dietary intakes of folate and vitamin B 6 were associated with reduced risk of mortality from stroke, coronary heart disease, and heart failure among Japanese. (Stroke. 2010;41:1285-1289.) Key Words: folate vitamin B coronary heart disease stroke follow-up study Folate, vitamin B 6, and vitamin B 12 are cofactors in homocysteine metabolism, and low intakes of these nutrients are associated with higher blood homocysteine concentrations, a potential risk factor for coronary heart disease (CHD) and stroke. 1 However, the effect of supplementation with these nutrients on secondary prevention of CHD or stroke has been controversial. 2,3 Furthermore, the evidence for an association between dietary intakes of folate and B vitamins and risk of cardiovascular disease (CVD) in Asian populations remains limited. No investigators have prospectively examined the associations of these vitamin intakes with heart failure, although blood homocysteine concentrations higher than the median ( 11.8 mmol/l for men and 11.1 mmol/l for women) were associated with a 2-fold increased risk. 4 There is thus an urgent need for a prospective study to replicate previous results in different populations and validate the associations. Subjects and Methods Study Population The Japan Collaborative Cohort Study began in 1988 to 1990. 5 The data from food frequency questionnaires were available for 24 386 men and 37 493 women age 40 to 79 years at baseline. We excluded persons who self-reported a history of CVD (n 2294) and cancer (n 855) at baseline because they were likely to change their dietary habits. The remaining 23 119 men and 35 611 women were enrolled in our study. The ethics committees of the Nagoya University School of Medicine and Osaka University Graduate School of Medicine approved the present study. Exposure Assessment and Mortality Surveillance The method for mortality surveillance has been described in detail elsewhere. 5 The underlying causes of death were determined according to the International Classification of Diseases, 10th revision, as follows: death from stroke (ICD I60-I69), CHD (ICD I20-I25), heart failure (ICD I50), and CVD (ICD I01-I99). Follow-up was conducted until the end of 2003, except for 4 communities, in which follow-up ended in 1999. The daily intake of nutrients for individuals was calculated by consulting the Standardized Tables of Food Composition, 5th ed. 6 Intakes of folate and vitamin B 6 were adjusted for total energy intake by means of a sex-specific residual model to reduce the influence of energy intake. Statistical Analysis The hazard ratios and their 95% CIs of mortality outcomes according to quintiles of dietary intakes of folate ( 272, 272 351, 352 430, 431 535, and 536 g/d), vitamin B 6 ( 0.79, 0.79 0.96, 0.97 1.11, 1.12 1.32, and 1.33 mg/d), and B 12 ( 4.5, 4.5 5.9, 6.0 7.6, 7.7 9.8, and 9.9 g/d) were calculated by using the Cox proportional-hazards model. Received January 11, 2010; final revision received February 2, 2010; accepted February 9, 2010. From Public Health (R.C., H.I.), Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka; Osaka Medical Center for Health Science and Promotion (R.C.), Osaka; Department of Food Sciences and Nutrition (C.D.), Faculty of Human Life and Environment, Nara Women s University, Nara; and Department of Public Health (S.K., A.T.), Aichi Medical University School of Medicine, Aichi, Japan. Correspondence to Prof Hiroyasu Iso, MD, Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871 Japan. E-mail iso@pbhel.med.osaka-u.ac.jp 2010 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.578906 1285

1286 Stroke June 2010 Table 1. Age-Adjusted Mean Values and Prevalence of CVD Risk Factors According to Quintiles of Dietary Folate, Vitamin B 6, and B 12 Intakes Folate, g/d Vitamin B 6, mg/d Vitamin B 12, g/d Quintile 1 (Low) 3 5 (High) Trend 1 (Low) 3 5 (High) Trend 1 (Low) 3 5 (High) Trend Men, median values 217 389 631 0.6 1.0 1.5 3.6 7.0 12.2 No. at risk 4623 4624 4624 4623 4624 4624 4623 4624 4624 Age, y 54.3 55.8 57.9 0.001 54.6 55.8 57.3 0.001 55.1 55.8 56.9 0.001 Ethanol intake, g/d 33.5 33.9 34.9 0.04 33.9 33.7 35.3 0.001 30.7 33.4 37.8 0.001 Current smoker, % 55 53 54 0.54 58 53 51 0.001 55 52 55 0.99 Body mass index, kg/m 2 22.7 22.7 22.7 0.53 22.6 22.7 22.8 0.001 22.6 22.8 22.7 0.002 History of hypertension, % 21 20 16 0.01 21 18 17 0.001 21 19 16 0.001 History of diabetes, % 7 6 5 0.001 7 7 5 0.001 7 6 5 0.001 Frequency of fish intake, times/wk 3 4 5 0.001 3 4 5 0.001 3 4 4 0.001 Saturated fatty acid, g/d 7 10 12 0.001 7 9 13 0.001 7 10 12 0.001 n-3 unsaturated acid, g/d 1.1 1.6 2.2 0.001 0.9 1.6 2.6 0.001 0.9 1.6 2.5 0.001 n-6 unsaturated acid, g/d 4.6 6.4 8.3 0.001 4.1 6.3 9.2 0.001 4.2 6.5 8.7 0.001 Intake of calories, kcal/d 1430 1751 2043 0.001 1299 1731 2211 0.001 1394 1739 2104 0.001 Women, median values 225 391 619 0.7 1.1 1.5 3.5 6.6 11.5 No. at risk 7122 7122 7122 7122 7122 7122 7122 7122 7122 Age, y 55.6 56.0 57.5 0.001 56.4 56.1 56.8 0.001 56.6 55.9 56.5 0.26 Ethanol intake, g/d 11.7 9.7 9.5 0.38 12.0 10.0 8.6 0.001 11.0 9.7 10.1 0.48 Current smoker, % 7 4 4 0.04 8 4 3 0.001 8 4 4 0.001 Body mass index, kg/m 2 23.0 22.9 23.0 0.09 22.9 22.9 23.0 0.20 22.9 22.9 23.0 0.07 History of hypertension, % 23 20 17 0.001 23 21 17 0.001 24 21 17 0.001 History of diabetes, % 4 4 3 0.41 5 3 2 0.001 4 3 3 0.002 Frequency of fish intake, times/wk 3 4 4 0.001 3 4 5 0.001 3 4 4 0.001 Saturated fatty acid, g/d 8 10 12 0.001 7 10 13 0.001 7 10 13 0.001 n-3 unsaturated acid, g/d 1.2 1.7 2.1 0.001 0.9 1.6 2.5 0.001 0.9 1.6 2.4 0.001 n-6 unsaturated acid, g/d 4.6 6.2 7.7 0.001 4.0 6.0 8.6 0.001 4.2 6.3 8.1 0.001 Intake of calories, kcal/d 1206 1445 1659 0.001 1095 1426 1799 0.001 1166 1442 1710 0.001 The confounding variables comprised age (year), body mass index (sex-specific quintiles), smoking status (never, ex-smoker, and current smoker of 1 19 and 20 cigarettes/d), alcohol intake category (never, ex-drinker, and current drinker of 1 22, 23 45, 46 68, and 69 g of ethanol per day), history of hypertension and diabetes (yes), as well as saturated fatty acids and n-3and n-6 polyunsaturated fatty acids (sex-specific quintiles). SAS version 9.1 (SAS Institute Inc, Cary, NC) was used to perform all statistical analyses (2 tailed). Results Table 1 shows baseline characteristics according to quintiles (lowest, middle, and highest) of dietary folate and vitamin B 6 and B 12 intakes. During the median 14-year follow-up, we documented 986 (500 in men and 486 in women) deaths from stroke, 424 (233 in men and 191 in women) from CHD, 318 (151 in men and 167 in women) from heart failure, and 2,087 (1066 in men and 1021 in women) from CVD. Dietary folate and vitamin B 6 intakes were inversely associated with mortality from heart failure for men and with mortality from stroke, CHD, and total CVD for women (Table 2). These inverse associations did not change materially after adjustment for CVD risk factors. When we excluded the subjects who were using multivitamin supplements (n 7334), the results did not change materially. For example, the multivariable hazard ratios (and 95% CIs) of CHD for the highest versus lowest quintiles were 0.62 (0.42 0.89) for folate, 0.51 (0.29 0.91) for vitamin B 6, and 1.35 (0.80 2.27) for vitamin B 12 intakes and those for heart failure were 0.76 (0.51 1.13) for folate, 0.60 (0.32 1.13) for vitamin B 6, and 1.57 (0.90 2.73) for vitamin B 12 (data not shown). Discussion We found inverse associations between folate and vitamin B 6 intakes and risk of mortality from stroke and CHD for Japanese, which are consistent with previous reports of these associations for Americans 7 and Europeans. 8 Furthermore, this study is the first to show that high dietary intakes of folate and vitamin B 6 were associated with a reduced risk of heart failure mortality for men. Mechanisms for these observed associations may involve the effects of these vitamin intakes on reductions of blood homocysteine concentrations. A meta-analysis of observational studies provided evidence that a 3- mol/l reduction in homocysteine level was associated with an 11% reduction in CHD risk and a 19% reduction in stroke risk. 1 A single, large, clinical trial of women, however, did not show any beneficial effect of

Cui et al Folate, B Vitamins, and Cardiovascular Disease 1287 Table 2. Sex-Specific Hazard Ratio (HRs) and 95% CIs of CVD According to Quintiles of Folate, Vitamin B 6, and B 12 Intake Men 1 (Low) 2 3 4 5 (High) Trend Total No. 4623 4624 4624 4624 4624 Folate Person-years 56 921 57 485 57 895 58 443 58 401 Stroke 81 91 93 115 120 Age-adjusted HR 1.00 1.04 (0.77 1.41) 0.96 (0.71 1.29) 1.15 (0.86 1.52) 1.05 (0.79 1.39) 0.74 Multivariable HR 1.00 1.06 (0.78 1.44) 1.01 (0.73 1.38) 1.19 (0.87 1.63) 1.12 (0.81 1.55) 0.50 CHD 54 43 39 52 45 Age-adjusted HR 1.00 0.74 (0.49 1.01) 0.61 (0.40 0.92) 0.78 (0.53 1.14) 0.60 (0.41 0.90) 0.01 Multivariable HR 1.00 0.79 (0.52 1.19) 0.69 (0.45 1.07) 0.92 (0.60 1.40) 0.72 (0.45 1.14) 0.16 Heart failure 31 34 34 29 23 Age-adjusted HR 1.00 1.02 (0.63 1.67) 0.93 (0.57 1.51) 0.77 (0.46 1.28) 0.54 (0.31 0.92) 0.02 Multivariable HR 1.00 1.08 (0.65 1.78) 1.03 (0.61 1.73) 0.80 (0.45 1.41) 0.50 (0.27 0.94) 0.03 CVD 190 197 197 244 238 Age-adjusted HR 1.00 0.96 (0.79 1.17) 0.87 (0.71 1.06) 1.04 (0.86 1.25) 0.89 (0.74 1.08) 0.24 Multivariable HR 1.00 1.00 (0.82 1.23) 0.94 (0.76 1.17) 1.12 (0.91 1.38) 0.97 (0.77 1.21) 0.75 Vitamin B 6 Person-years 55 793 56 872 58 008 59 072 59 404 Stroke 87 91 104 107 111 Age-adjusted HR 1.00 0.96 (0.71 1.28) 1.01 (0.76 1.34) 0.99 (0.75 1.31) 0.95 (0.72 1.26) 0.74 Multivariable HR 1.00 0.95 (0.68 1.32) 1.14 (0.78 1.66) 1.19 (0.77 1.82) 1.08 (0.65 1.78) 0.77 CHD 58 46 41 42 46 Age-adjusted HR 1.00 0.72 (0.49 1.06) 0.60 (0.40 0.89) 0.58 (0.39 0.86) 0.59 (0.40 0.87) 0.001 Multivariable HR 1.00 0.65 (0.41 1.01) 0.52 (0.30 0.88) 0.54 (0.29 0.98) 0.64 (0.32 1.30) 0.22 Heart failure 36 24 35 32 24 Age-adjusted HR 1.00 0.62 (0.37 1.04) 0.87 (0.55 1.38) 0.75 (0.46 1.20) 0.53 (0.31 0.89) 0.02 Multivariable HR 1.00 0.71 (0.39 1.29) 0.96 (0.49 1.89) 0.73 (0.34 1.60) 0.39 (0.15 1.00) 0.05 CVD 210 192 211 222 231 Age-adjusted HR 1.00 0.83 (0.69 1.01) 0.85 (0.70 1.03) 0.85 (0.70 1.03) 0.82 (0.68 0.99) 0.04 Multivariable HR 1.00 0.85 (0.68 1.07) 0.92 (0.71 1.20) 0.96 (0.72 1.29) 0.93 (0.66 1.30) 0.66 Vitamin B 12 Person-years 55 945 57 141 58 601 58 930 58 533 Stroke 80 104 104 82 130 Age-adjusted HR 1.00 1.26 (0.94 1.68) 1.15 (0.86 1.54) 0.86 (0.63 1.17) 1.38 (1.05 1.83) 0.02 Multivariable HR 1.00 1.21 (0.85 1.71) 1.36 (0.91 2.02) 1.03 (0.66 1.62) 1.59 (1.01 2.52) 0.05 CHD 50 50 44 47 42 Age-adjusted HR 1.00 0.95 (0.64 1.41) 0.77 (0.52 1.16) 0.78 (0.52 1.16) 0.71 (0.47 1.06) 0.10 Multivariable HR 1.00 0.93 (0.58 1.49) 0.81 (0.46 1.43) 0.90 (0.49 1.67) 0.93 (0.48 1.80) 0.83 Heart failure 28 34 31 28 30 Age-adjusted HR 1.00 1.19 (0.72 1.97) 1.02 (0.61 1.70) 0.88 (0.52 1.48) 0.95 (0.57 1.60) 0.86 Multivariable HR 1.00 1.72 (0.95 3.14) 1.61 (0.79 3.29) 1.53 (0.69 3.36) 1.53 (0.65 3.46) 0.34 CVD 191 215 209 208 243 Age-adjusted HR 1.00 1.08 (0.89 1.32) 0.97 (0.79 1.18) 0.91 (0.75 1.11) 1.08 (0.89 1.30) 0.43 Multivariable HR 1.00 1.16 (0.92 1.47) 1.20 (0.91 1.58) 1.20 (0.89 1.62) 1.43 (0.89 1.95) 0.03 Multivariable adjustment on age, body mass index, history of hypertension and diabetes, smoking status, ethanol and energy intakes, as well as intakes of saturated fatty acids and n-3 and n-6 polyunsaturated fatty acids.

1288 Stroke June 2010 Table 2. Continued Women 1 (Low) 2 3 4 5 (High) 7122 7122 7122 7122 7122 Trend 89 723 90 439 91 288 91 784 92 778 110 95 90 87 104 1.00 0.92 (0.70 1.21) 0.81 (0.61 1.07) 0.74 (0.56 0.98) 0.82 (0.63 1.07) 0.14 1.00 0.93 (0.70 1.23) 0.81 (0.60 1.09) 0.74 (0.55 1.01) 0.83 (0.61 1.12) 0.22 54 44 21 44 28 1.00 0.89 (0.60 1.32) 0.40 (0.24 0.64) 0.77 (0.52 1.15) 0.46 (0.29 0.72) 0.001 1.00 0.97 (0.64 1.46) 0.43 (0.26 0.73) 0.89 (0.58 1.37) 0.57 (0.34 0.96) 0.03 37 32 31 29 38 1.00 0.95 (0.59 1.53) 0.85 (0.53 1.37) 0.76 (0.47 1.23) 0.92 (0.59 1.45) 0.72 1.00 1.00 (0.61 1.62) 0.92 (0.56 1.53) 0.83 (0.49 1.41) 1.03 (0.61 1.75) 0.91 245 189 179 200 208 1.00 0.80 (0.69 1.00) 0.73 (0.60 0.88) 0.77 (0.64 0.93) 0.74 (0.61 0.89) 0.001 1.00 0.86 (0.71 1.05) 0.77 (0.63 0.94) 0.83 (0.67 1.01) 0.83 (0.67 1.03) 0.08 87 482 89 596 91 447 92 705 94 781 115 108 79 93 91 1.00 1.01 (0.78 1.32) 0.71 (0.54 0.95) 0.83 (0.63 1.09) 0.79 (0.60 1.04) 0.09 1.00 0.88 (0.64 1.20) 0.56 (0.39 0.81) 0.62 (0.41 0.93) 0.63 (0.39 1.03) 0.06 66 32 37 26 30 1.00 0.53 (0.35 0.81) 0.59 (0.39 0.88) 0.41 (0.26 0.64) 0.46 (0.30 0.71) 0.001 1.00 0.48 (0.29 0.77) 0.55 (0.32 0.94) 0.40 (0.20 0.77) 0.47 (0.21 1.04) 0.06 42 33 29 35 28 1.00 0.87 (0.55 1.38) 0.74 (0.46 1.19) 0.89 (0.57 1.40) 0.71 (0.44 1.15) 0.16 1.00 0.97 (0.57 1.65) 0.86 (0.46 1.63) 1.01 (0.50 2.05) 0.86 (0.37 2.02) 0.73 259 208 191 188 175 1.00 0.87 (0.73 1.05) 0.77 (0.64 0.93) 0.75 (0.62 0.90) 0.68 (0.56 0.82) 0.001 1.00 0.84 (0.68 1.04) 0.73 (0.57 0.94) 0.73 (0.55 0.97) 0.73 (0.52 1.02) 0.06 87 605 90 087 92 094 92 768 93 458 106 99 91 105 85 1.00 1.01 (0.77 1.33) 0.94 (0.71 1.33) 1.06 (0.81 1.38) 0.86 (0.65 1.15) 0.32 1.00 1.03 (0.73 1.44) 0.99 (0.66 1.48) 1.25 (0.82 1.92) 1.13 (0.71 1.79) 0.61 52 38 42 28 31 1.00 0.80 (0.53 1.22) 0.90 (0.60 1.35) 0.59 (0.37 0.93) 0.66 (0.42 1.04) 0.07 1.00 0.97 (0.59 1.61) 1.71 (0.94 3.11) 1.18 (0.60 2.35) 1.39 (0.67 2.89) 0.37 43 31 31 30 32 1.00 0.80 (0.50 1.27) 0.82 (0.51 1.30) 0.78 (0.49 1.24) 0.85 (0.54 1.34) 0.48 1.00 1.02 (0.59 1.77) 1.31 (0.67 2.57) 1.38 (0.68 2.81) 1.79 (0.85 3.76) 0.13 244 210 195 189 183 1.00 0.94 (0.78 1.13) 0.88 (0.73 1.06) 0.83 (0.69 1.01) 0.82 (0.67 0.99) 0.04 1.00 1.01 (0.81 1.26) 1.11 (0.85 1.45) 1.21 (0.91 1.62) 1.33 (0.97 1.81) 0.07

Cui et al Folate, B Vitamins, and Cardiovascular Disease 1289 folic acid supplementation on risk of CVD. 2 A more recent clinical trial of men and women has demonstrated that the lowering of homocysteine by supplementation with folic acid, vitamin B 6, and vitamin B 12 reduced the risk of stroke. 3 Taken together, our results suggest that dietary intakes of folate and vitamin B 6 may be useful to prevent CVD. The supplementation with folate and vitamin B 6 was not taken into account in the present study, but the observed associations did not change after exclusion of persons who were taking multivitamin supplements. Also, we used the mortality data as end points, which may have led to misclassification in the diagnosis of stroke, CHD, and heart failure. However, previous validation studies indicated the validity of death certificate diagnoses for these outcomes because of the widespread use of computed tomography or magnetic resonance imaging for stroke diagnosis 9 and of ECG and cardiac enzyme examinations for CHD and heart failure. 10 In summary, high dietary folate and vitamin B 6 intakes were associated with a reduced risk of mortality from stroke, CHD, and heart failure among Japanese. Acknowledgments The authors thank all members of the Japan Collaborative Cohort Study, which is available in detail at: http://www.aichi-med-u.ac.jp/ jacc/member.html. Sources of Funding This study was supported by the Ministry of Education, Science, Sports and Culture of Japan (Monbusho) and the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Monbu-kagakusho): grant numbers 61010076, 62010074, 63010074, 1010068, 2151065, 3151064, 4151063, 5151069, 6279102, 11181101, 18014011, 20014026, 17015022, 18014011, 14207019 and 19390174. None. Disclosures References 1. Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. J Am Med Assoc. 2002;288: 2015 2022. 2. Albert CM, Cook NR, Gaziano JM, Zaharris E, MacFadyen J, Danielson E, Buring JE, Manson JE. Effect of folic acid and B vitamins on risk of cardiovascular events and total mortality among women at high risk for cardiovascular disease: a randomized trial. J Am Med Assoc. 2008;299: 2027 2036. 3. Saposnik G, Ray JG, Sheridan P, McQueen M, Lonn E; Heart Outcomes Prevention Evaluation 2 Investigators. Homocysteine-lowering therapy and stroke risk, severity, and disability: additional findings from the HOPE 2 trial. Stroke. 2009;40:1365 1372. 4. Vasan RS, Beiser A, D Agostino RB, Levy D, Selhub J, Jacques PF, Rosenberg IH, Wilson PW. Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction. JAmMed Assoc. 2003;289:1251 1257. 5. Ohno Y, Tamakoshi A, and the Journal of the American College of Cardiology Study Group. Japan Collaborative Cohort Study for evaluation of cancer risk sponsored by Monbusho (J Am Coll Cardiol study). J Epidemiol. 2001;11:144 150. 6. Science and Technology Agency. Standard Tables of Food Composition in Japan, 5th revised ed. Tokyo: Printing Bureau, Ministry of Finance; 2002. 7. Rimm E, Willett W, Hu F, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ. Folate and vitamin B 6 from diet and supplements in relation to risk of coronary heart disease among women. J Am Med Assoc. 1998;279:359 364. 8. Voutilainen S, Rissanen T, Virtanen J, Lakka T, Salonen J. Low dietary folate intake is associated with an excess incidence of acute coronary events: the Kuopio Ischemic Heart Disease Risk Factor Study. Circulation. 2001;103:2674 2680. 9. Kita Y, Okayama A, Ueshima H, Wada M, Nozaki A, Choudhury SR, Bonita R, Inamoto Y, Kasamatsu T. Stroke incidence and case fatality in Shiga, Japan, 1989 1993. Int J Epidemiol. 1999;28:1059 1065. 10. Baba S, Ozawa H, Sakai Y, Terao A, Konishi M, Tatara K. Heart disease deaths in a Japanese urban area evaluated by clinical and police records. Circulation. 1994;89:109 115.