cerebral infarction; diet; folic acid; methionine; stroke; vitamin B 6; vitamin B 12

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1 American Journal of Epidemiology ª The Author Published by the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please Vol. 167, No. 8 DOI: /aje/kwm395 Advance Access publication February 12, 2008 Original Contribution Folate, Vitamin B 6, Vitamin B 12, and Methionine Intakes and Risk of Stroke Subtypes in Male Smokers Susanna C. Larsson 1, Satu Männistö 2, Mikko J. Virtanen 2, Jukka Kontto 2, Demetrius Albanes 3, and Jarmo Virtamo 2 1 National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 2 Department of Health Promotion and Chronic Disease Prevention, National Public Health Institute, Helsinki, Finland. 3 National Cancer Institute, National Institutes of Health, Bethesda, MD. Received for publication September 8, 2007; accepted for publication December 13, The associations of dietary folate, vitamin B 6, vitamin B 12, and methionine intakes with risk of stroke subtypes were examined among 26,556 male Finnish smokers, aged years, enrolled in the Alpha-Tocopherol, Beta- Carotene Cancer Prevention Study. Dietary intake was assessed at baseline by using a validated food frequency questionnaire. During a mean follow-up of 13.6 years, from 1985 through 2004, 2,702 cerebral infarctions, 383 intracerebral hemorrhages, and 196 subarachnoid hemorrhages were identified from national registers. In analyses adjusting for age and cardiovascular risk factors, a high folate intake was associated with a statistically significant lower risk of cerebral infarction but not intracerebral or subarachnoid hemorrhages. The multivariate relative risk of cerebral infarction was 0.80 (95% confidence interval: 0.70, 0.91; p trend ¼ 0.001) for men in the highest versus lowest quintile of folate intake. Vitamin B 6, vitamin B 12, and methionine intakes were not significantly associated with any subtype of stroke. These findings in men suggest that a high dietary folate intake may reduce the risk of cerebral infarction. cerebral infarction; diet; folic acid; methionine; stroke; vitamin B 6; vitamin B 12 Abbreviations: CI, confidence interval; HDL, high density lipoprotein; ICD, International Classification of Diseases. Substantial evidence has accrued linking elevated blood total homocysteine concentrations to increased risk of ischemic stroke (1 4). Furthermore, many epidemiologic studies have found that individuals homozygous for the T allele of the methylenetetrahydrofolate reductase C677T polymorphism have higher plasma homocysteine concentrations and increased risk of stroke compared with those with the CC genotype, providing further support for a causal relation between homocysteine and stroke (5, 6). The B vitamins folate, vitamin B 6 (pyridoxine), and vitamin B 12 (cobalamin) are important regulators of homocysteine metabolism in the body, and randomized controlled trials have demonstrated that supplementation with folate (natural dietary folate or the synthetic folic acid) alone or in combination with vitamins B 6 and B 12 significantly reduces blood homocysteine concentrations (7 10). Although increased intakes of these B vitamins could plausibly reduce the risk of stroke, findings from observational studies on folate (11 17), vitamin B 6 (14), and vitamin B 12 (14, 15, 17) in relation to stroke risk have been inconsistent. Likewise, randomized clinical trials examining the effects of supplemental folic acid and other B vitamins on stroke incidence among individuals with preexisting cardiovascular or renal disease have produced conflicting results (18, 19). Methionine is a sulfur-containing amino acid naturally found in the diet. It is the precursor of S-adenosylmethionine, which forms homocysteine following the removal of the methyl group. When methionine intake is high, the Correspondence to Dr. Susanna C. Larsson, Division of Nutritional Epidemiology, National Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, SE Stockholm, Sweden ( susanna.larsson@ki.se). 954

2 Dietary Intakes and Stroke Risk in Male Smokers 955 formation of homocysteine increases, leading to increased blood homocysteine concentrations (20). To our knowledge, no previous study has evaluated the relation between methionine intake and risk of stroke. The purpose of the present study was to examine prospectively the associations of dietary folate, vitamin B 6, vitamin B 12, and methionine intakes with risk of stroke among Finnish male smokers enrolled in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. MATERIALS AND METHODS Study cohort The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study was established between 1985 and 1988 when 29,133 male smokers aged years and living in southwestern Finland were recruited to participate in a prevention trial (21). The study was a randomized, double-blinded, placebocontrolled, prevention trial undertaken to determine whether supplementation with alpha-tocopherol (50 mg/day), betacarotene (20 mg/day), or both could reduce the incidence of lung and other cancers. At baseline, men were excluded from the trial if they smoked fewer than five cigarettes per day; had a history of cancer, severe angina on exertion, chronic renal insufficiency, liver cirrhosis, chronic alcoholism, or other medical conditions that might limit long-term participation; received anticoagulant therapy; or used vitamin E, vitamin A, or beta-carotene supplements in excess of predefined doses. The trial continued until April 1993, with the cohort followed up through national registers thereafter. The present analysis is based on 26,556 participants with complete baseline dietary data and without history of stroke. Written, informed consent was obtained from each participant before randomization. The study was approved by the institutional review boards of the National Public Health Institute of Finland and the US National Cancer Institute. Baseline data collection At baseline, the participants completed questionnaires on general characteristics as well as medical, smoking, and physical activity histories (21). Height, weight, and blood pressure were measured. A blood sample was drawn, and serum was stored at 70 C. Serum total cholesterol and high density lipoprotein (HDL) cholesterol levels were determined enzymatically (cholesterol oxidase-p-aminophenazone (CHOD-PAP) method; Boehringer Ingelheim GmbH, Ingelheim, Germany). Serum pepsinogen I was measured from the baseline samples of 22,333 men (22). Dietary intake was assessed with a validated selfadministered food frequency questionnaire that included 276 food items and mixed dishes commonly consumed in Finland (23). It was used with a portion-size color picture booklet of 122 photographs of foods, each with 3 5 different portion sizes. Participants were asked to report their average consumption and portion size for each food/dish during the previous year. Frequencies were reported as the number of times per month, week, or day. At the first visit, the questionnaire, together with the picture booklet, was given to the participant to be filled in at home. At the second baseline visit, 2 weeks later, the questionnaire was returned, and a trained nurse checked and completed it, spending on average 30 minutes interviewing the participant about possible discrepancies. Thereafter, a senior nutritionist reviewed centrally the questionnaire and decided on final approval. The questionnaire was rejected if the participant had not been involved in filling it, or if the consumption of certain foods exceeded preset reasonable limits. The questionnaire was satisfactorily completed by 27,111 participants (93 percent). Intake of nutrients was calculated by use of the food composition database at the National Public Health Institute in Finland. The dietary method was validated in a pilot study carried out among 189 men before the study (23). The men completed the food frequency questionnaire at the beginning and end of a 6-month period, and they kept food consumption records for 24 days ( days) in the interim period. The correlations comparing the first questionnaire with food records were 0.6 for folate, 0.5 for vitamin B 6, 0.4 for vitamin B 12, and 0.5 for methionine (unpublished data). Ascertainment of stroke The study endpoint was first-ever stroke that occurred between the date of randomization and December 31, The strokes were further divided into cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage, and unspecified stroke. The endpoints were identified by record linkage with the National Hospital Discharge Register and the National Register of Causes of Death. Both registers used the codes of the International Classification of Diseases (ICD): The Eighth Revision (ICD-8) was used until the end of 1986, the Ninth Revision (ICD-9) through the end of 1996, and the Tenth Revision (ICD-10) thereafter. The endpoints comprised ICD-8 codes and 436, ICD-9 codes , , and 436, and ICD-10 codes I60, I61, I63, and I64, excluding ICD-8 codes and representing subdural hemorrhage and ICD-9 codes 4330X, 4331X, 4339X, and 4349X denoting occlusion or cerebral or precerebral artery stenosis without cerebral infarction. In a reviewed sample, the diagnoses of cerebral infarction, subarachnoid hemorrhage, and intracerebral hemorrhage proved correct by strict preset criteria (24, 25) in 90 percent, 79 percent, and 82 percent of the discharge diagnoses and in 92 percent, 95 percent, and 91 percent of the causes of death, respectively (26). Statistical analysis For each participant, the person-time of follow-up was counted from the date of randomization to the date of the first stroke, death, or the end of follow-up (December 31, 2004), whichever occurred first. When the risk estimates for a subtype of stroke were analyzed, the other subtypes were treated as censored. All nutrients were energy adjusted by the residual method (27). Participants were categorized into quintiles according to their dietary intakes of folate, vitamin B 6, vitamin B 12, and methionine. Relative risks and 95 percent

3 956 Larsson et al. TABLE 1. Baseline characteristics of 26,556 men in by lowest and highest quintile of dietary folate, vitamin B 6, vitamin B 12, and methionine intake, Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study* Folate Vitamin B 6 Vitamin B 12 Methionine confidence intervals were calculated with the Cox proportional hazards model (28). All models were controlled for age at baseline and supplementation group (alpha-tocopherol, beta-carotene, both, or placebo). In multivariate models, we further adjusted for cardiovascular risk factors (number of cigarettes smoked daily, body mass index, systolic and diastolic blood pressures, serum total cholesterol, serum HDL cholesterol, histories of diabetes and coronary heart disease, leisure-time physical activity, and alcohol consumption) and total energy intake. Tests for trends were performed by assigning the median value to each quintile and treating this variable as a continuous variable. Effect modification was examined by stratified analyses, and the significance of interactions was tested by including a cross-product term of the nutrient intake (as a continuous variable) and the covariate of interest (as a dichotomous variable). The statistical analyses were performed with Stata, version 9.2, software (Stata Corporation, College Station, Texas). All p values are two sided, and p < 0.05 was considered statistically significant. RESULTS Baseline characteristics of the study population according to dietary intakes of folate, vitamin B 6, vitamin B 12, and methionine are presented in table 1. On average, men with higher folate intake were slightly younger, smoked less cigarettes daily, had higher body mass index, had lower systolic blood pressure and lower serum total and HDL cholesterol, Quintile 1 Quintile 5 Quintile 1 Quintile 5 Quintile 1 Quintile 5 Quintile 1 Quintile 5 Age at baseline (years) Smoking (cigarettes/day) Body mass index (kg/m 2 ) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Serum total cholesterol (mmol/liter) Serum HDLy cholesterol (mmol/liter) Diabetes (%) History of coronary heart disease (%)z Physically active (%) Daily intake Energy (kcal) 2,647 2,645 2,694 2,673 2,638 2,648 2,668 2,644 Alcohol intake (g) Folate (lg) Vitamin B 6 (mg) Vitamin B 12 (lg) Methionine (mg) 1,892 2,211 1,797 2,229 1,740 2,229 1,554 2,514 * All values other than age at baseline were standardized to the age distribution of the cohort. y HDL, high density lipoprotein. z Including myocardial infarction and angina pectoris. Moderate or heavy activity at leisure time. were more likely to have a history of diabetes or coronary heart disease, were more likely to be physically active, and consumed less alcohol. Men with higher intakes of vitamin B 6 and methionine showed characteristics similar to those who had higher folate intake. Compared with men with low vitamin B 12 intake, those with higher intake tended to smoke more cigarettes, have higher body mass index, have higher systolic and diastolic blood pressure levels, have higher serum total cholesterol, be more likely to have a history of diabetes or coronary heart disease, be physically active, and consume more alcohol. During a mean follow-up of 13.6 years (360,187 personyears), 2,702 cerebral infarctions, 383 intracerebral hemorrhages, 196 subarachnoid hemorrhages, and 84 unspecified strokes occurred. Dietary folate intake was statistically significantly inversely associated with the risk of cerebral infarction but not intracerebral or subarachnoid hemorrhage after adjustment for age and supplementation group and, in multivariate models, further adjusted for cigarettes/day, body mass index, systolic and diastolic blood pressure, serum total and HDL cholesterol, histories of diabetes and coronary heart disease, leisure-time physical activity, and alcohol and total energy intakes (table 2). The multivariate relative risk of cerebral infarction for men in the highest quintile of folate intake compared with those in the lowest quintile was 0.80 (95 percent confidence interval (CI): 0.70, 0.91). The association remained after further adjustment for dietary fiber, magnesium, and saturated fat intakes (relative risk ¼ 0.82, 95 percent CI: 0.71, 0.96). The crude incidence rates of

4 Dietary Intakes and Stroke Risk in Male Smokers 957 cerebral infarction were 82.0 and 60.7 cases per 10,000 persons per year in the lowest and the highest quintile of dietary folate intake. Vitamin B 6 intake was statistically significantly inversely associated with the risk of cerebral infarction after adjustment for age and supplementation group only, but this association was attenuated and not statistically significant after adjustment for cardiovascular risk factors. We found no clear association of vitamin B 12 or methionine intake with any subtype of stroke (table 2). The null results for dietary vitamin B 12 persisted when we excluded participants with low serum pespinogen I level (<25 lg/liter). The relation between dietary folate intake and the risk of cerebral infarction was statistically significantly modified by dietary vitamin B 12 intake (p interaction ¼ 0.01). The multivariate relative risks of cerebral infarction comparing men in the highest versus the lowest quintile of folate intake were 0.72 (95 percent CI: 0.59, 0.87) among those with a high vitamin B 12 intake (above the median, i.e., 10.2 lg/day) and 0.86 (95 percent CI: 0.71, 1.03) among those with a low vitamin B 12 intake. The results for folate presented in table 2 were similar when we added to the multivariate model the interaction term between folate and vitamin B 12 intake. Despite high dietary intake of vitamin B 12, the serum vitamin B 12 concentration may be low because of reduced absorption caused by atrophic gastric mucosa. We had no data on serum vitamin B 12, but we used the serum pepsinogen I level as a proxy of vitamin B 12 absorption capacity. The association between folate intake and risk of cerebral infarction did not vary significantly by the absorption capacity of vitamin B 12 ; the multivariate relative risks for the highest versus the lowest quintile of folate intake were 0.86 (95 percent CI: 0.74, 1.00) among participants with normal absorption (serum pepsinogen I 25 lg/liter) and 0.69 (95 percent CI: 0.42, 1.11) among those with reduced absorption (serum pepsinogen I <25 lg/liter). The association between folate intake and risk of cerebral infarction was also statistically significantly modified by history of coronary heart disease at baseline (p interaction ¼ 0.03) but not by other cardiovascular risk factors (alcohol consumption, cigarettes/day, smoking years, body mass index, hypertension, serum total cholesterol, serum HDL cholesterol, physical activity) or supplementation group. The multivariate relative risks of cerebral infarction comparing men in the highest versus the lowest quintile of folate intake were 0.74 (95 percent CI: 0.65, 0.86) among men with no history of coronary heart disease and 1.16 (95 percent CI: 0.84, 1.66) among those with a history of coronary heart disease. There was no statistically significant interaction between vitamin B 6, vitamin B 12, or methionine intake and any cardiovascular risk factor. To address the potential for increased exposure misclassification over time, we divided the follow-up time into 1 10 years and years. The relations of folate, vitamin B 6, vitamin B 12, and methionine intakes with stroke risk did not differ materially by follow-up time. DISCUSSION In this large cohort of male Finnish smokers, a high dietary folate intake was associated with a significantly lower risk of cerebral infarction. Major food sources of folate in this population were rye (contributing 17 percent of folate intake), potatoes (9 percent), vegetables (8 percent), egg (7 percent), and wheat (5 percent). Vitamin B 6, vitamin B 12, and methionine intakes were not significantly associated with risk of any stroke subtype after adjustment for potential confounders. There was a nonsignificant inverse relation between folate intake and risk of intracerebral hemorrhage. The relative risk of intracerebral hemorrhage for men in the highest versus the lowest quintile of folate intake was comparable to the corresponding relative risk for cerebral infarction. The lack of a statistically significant association between folate intake and intracerebral hemorrhage may be due to inadequate power to detect this modest association. An inverse association between folate intake and risk of stroke is biologically plausible because folic acid supplementation lowers blood homocysteine (7 10), which in high concentrations may cause vascular damage (endothelial dysfunction, increased arterial intimal-medial thickness, and increased arterial stiffness) and increased procoagulant activity (29). Meta-analyses have estimated that a 3-lmol/ liter decrease in blood homocysteine concentrations is associated with a percent lower risk of stroke (1, 4). In a recent meta-analysis of eight randomized, controlled trials, consisting of 16,841 individuals with preexisting vascular disease, the overall relative risk of stroke for patients receiving folic acid supplementation (with or without combination with vitamins B 6 and B 12 ) compared with controls was 0.82 (95 percent CI: 0.68, 1.00; p ¼ 0.045) (19). A greater beneficial effect was seen in those trials with a treatment duration of >36 months, a decrease in homocysteine concentration of >20 percent, no fortification of grains, and no history of stroke (19). Another meta-analysis of the same eight randomized trials found an overall relative risk of 0.86 (95 percent CI: 0.71, 1.04) (18). Our findings for dietary folate are not comparable to those from randomized trials, because folate derived from foods is different in type and amount from that derived from supplementation. Our observed inverse association between folate intake and risk of cerebral infarction is consistent with results from most previous prospective studies. Folate intake was statistically significantly inversely associated with the risk of ischemic stroke in the Health Professionals Follow-up Study (14) and with total stroke in the First National Health and Nutrition Examination Survey Epidemiologic Follow-up Study (12). In addition, two prospective studies on serum or plasma folate found a statistically significant lower risk of stroke for individuals in the highest versus lowest category of blood folate (15, 16). The Nurses Health Study (13) and the Bronx Longitudinal Aging Study (17) reported no association between folate intake or serum folate concentration, respectively, and risk of stroke. In the present study, the inverse association between folate intake and cerebral infarction appeared to be confined to men with no history of coronary heart disease at baseline. This may be due to the facts that men with coronary heart disease have higher serum homocysteine concentrations (30, 31) and that folate intake among those men may not be sufficient to protect against stroke.

5 958 Larsson et al. TABLE 2. Relative risks of stroke subtypes by quintiles of dietary folate, vitamin B 6, vitamin B 12, and methionine intake among 26,556 men, Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, Nutrient Quintile 1 (lowest) Prospective data on vitamin B 6 and vitamin B 12 in relation to risk of stroke are limited. In the Health Professionals Follow-up Study, no association was found between vitamin B 6 intake and stroke (14). However, men in the highest quintile of vitamin B 12 intake had a statistically nonsignificant lower risk of ischemic stroke compared with men in the lowest quintile (relative risk ¼ 0.73, 95 percent CI: 0.52, 1.03) (14). Serum or plasma vitamin B 12 concentrations were not related to risk of stroke in the Northern Sweden Health and Disease Cohort (15) or the Bronx Longitudinal Aging Study (17). Hence, our null findings for vitamin B 6 and vitamin B 12 intakes are generally consistent with previous studies. The absorption of vitamin B 12 is dependent on the intrinsic factor secreted by the healthy gastric mucosa. Intrinsic Nutrient intake Quintile 2 Quintile 3 Quintile 4 Quintile 5 (highest) Folate Median intake (lg/day) No. of person-years 68,639 71,263 72,725 72,819 74,741 Cerebral infarction No. of cases Relative risk (95% CI*)y (0.84, 1.06) 1.03 (0.91, 1.15) 0.91 (0.81, 1.02) 0.76 (0.67, 0.86) <0.001 Relative risk (95% CI)z (0.88, 1.11) 1.09 (0.97, 1.23) 0.98 (0.87, 1.10) 0.80 (0.70, 0.91) Intracerebral hemorrhage No. of cases Relative risk (95% CI)y (0.72, 1.31) 0.85 (0.63, 1.16) 0.73 (0.53, 1.01) 0.81 (0.59, 1.11) 0.07 Relative risk (95% CI)z (0.75, 1.38) 0.89 (0.65, 1.23) 0.77 (0.55, 1.07) 0.83 (0.60, 1.16) 0.11 Subarachnoid hemorrhage No. of cases Relative risk (95% CI)y (0.90, 2.20) 0.94 (0.58, 1.53) 1.32 (0.84, 2.07) 1.13 (0.71, 1.81) 0.79 Relative risk (95% CI)z (0.91, 2.25) 0.94 (0.57, 1.55) 1.33 (0.84, 2.12) 1.15 (0.71, 1.87) 0.77 Vitamin B 6 Median intake (mg/day) No. of person-years 68,674 71,186 72,434 73,710 74,183 Cerebral infarction No. of cases Relative risk (95% CI)y (0.87, 1.10) 0.88 (0.78, 0.99) 0.91 (0.81, 1.03) 0.88 (0.78, 1.00) 0.02 Relative risk (95% CI)z (0.91, 1.15) 0.90 (0.80, 1.01) 0.95 (0.84, 1.07) 0.91 (0.80, 1.03) 0.06 Intracerebral hemorrhage No. of cases Relative risk (95% CI)y (0.66, 1.22) 0.84 (0.62, 1.15) 0.78 (0.57, 1.07) 0.83 (0.61, 1.14) 0.17 Relative risk (95% CI)z (0.70, 1.29) 0.88 (0.64, 1.21) 0.83 (0.60, 1.14) 0.90 (0.65, 1.24) 0.39 Subarachnoid hemorrhage No. of cases Relative risk (95% CI)y (0.58, 1.38) 0.74 (0.47, 1.17) 0.81 (0.52, 1.26) 0.98 (0.65, 1.50) 0.90 Relative risk (95% CI)z (0.58, 1.40) 0.73 (0.46, 1.16) 0.82 (0.52, 1.29) 1.00 (0.65, 1.54) 0.97 Table continues p trend factor secretion is low from atrophic gastric mucosa resulting in negligible absorption of vitamin B 12 despite a normal dietary supply. Hence, a possible relation between vitamin B 12 intake and risk of stroke may be attenuated toward unity in subjects with atrophic gastritis. We therefore excluded from the analyses men with a low serum pepsinogen I level and thus atrophic gastritis but still found no association between vitamin B 12 intake and stroke risk. The lack of observed association between vitamin B 12 intake and the risk of stroke may not be surprising given that vitamin B 12 has a relatively small effect on homocysteine concentrations (8), and only a minor proportion of study participants had vitamin B 12 deficiency (32), being usually related to a problem of absorption (due to atrophic gastritis) rather than nutrition. We found, however, that

6 Dietary Intakes and Stroke Risk in Male Smokers 959 TABLE 2. Continued Nutrient intake Nutrient Quintile 1 (lowest) Quintile 2 Quintile 3 Quintile 4 Quintile 5 (highest) p trend Vitamin B 12 Median intake (lg/day) No. of person-years 72,935 71,533 71,686 71,805 72,228 Cerebral infarction No. of cases Relative risk (95% CI)y (0.90, 1.14) 1.03 (0.92, 1.16) 1.02 (0.90, 1.15) 1.05 (0.93, 1.18) 0.44 Relative risk (95% CI)z (0.89, 1.13) 1.01 (0.89, 1.13) 1.01 (0.89, 1.14) 1.01 (0.90, 1.14) 0.82 Intracerebral hemorrhage No. of cases Relative risk (95% CI)y (0.51, 0.96) 0.78 (0.57, 1.07) 0.78 (0.57, 1.06) 1.05 (0.79, 1.40) 0.34 Relative risk (95% CI)z (0.51, 0.97) 0.79 (0.58, 1.08) 0.79 (0.58, 1.08) 1.05 (0.78, 1.40) 0.34 Subarachnoid hemorrhage No. of cases Relative risk (95% CI)y (0.54, 1.39) 1.15 (0.74, 1.78) 1.02 (0.65, 1.59) 1.19 (0.78, 1.84) 0.30 Relative risk (95% CI)z (0.53, 1.35) 1.12 (0.72, 1.73) 0.99 (0.63, 1.56) 1.17 (0.76, 1.80) 0.34 Methionine Median intake (mg/day) 1,598 1,831 1,996 2,163 2,445 No. of person-years 71,518 72,140 72,528 72,908 71,093 Cerebral infarction No. of cases Relative risk (95% CI)y (0.86, 1.09) 1.06 (0.94, 1.19) 0.94 (0.83, 1.06) 1.06 (0.94, 1.19) 0.44 Relative risk (95% CI)z (0.89, 1.14) 1.11 (0.98, 1.25) 0.99 (0.87, 1.12) 1.05 (0.93, 1.19) 0.56 Intracerebral hemorrhage No. of cases Relative risk (95% CI)y (0.55, 1.02) 0.68 (0.49, 0.93) 0.71 (0.52, 0.97) 0.99 (0.74, 1.32) 0.92 Relative risk (95% CI)z (0.58, 1.08) 0.72 (0.52, 1.01) 0.77 (0.56, 1.06) 1.06 (0.78, 1.43) 0.69 Subarachnoid hemorrhage No. of cases Relative risk (95% CI)y (0.78, 1.95) 0.84 (0.51, 1.39) 1.42 (0.91, 2.21) 1.39 (0.89, 2.18) 0.10 Relative risk (95% CI)z (0.79, 2.01) 0.86 (0.52, 1.44) 1.49 (0.94, 2.35) 1.49 (0.93, 2.37) 0.06 * CI, confidence interval. y Adjusted for age and supplementation group. z Adjusted for age, supplementation group, total number of cigarettes smoked daily, body mass index, systolic and diastolic blood pressure, serum total cholesterol, serum high density lipoprotein cholesterol, histories of diabetes and coronary heart disease, leisure-time physical activity, and intakes of alcohol and total energy. folate intake was more strongly inversely related to cerebral infarction in men with a high vitamin B 12 intake. A metaanalysis of randomized trials estimated that folic acid supplementation could be expected to reduce homocysteine concentrations by percent (depending on dose) and that vitamin B 12 produced 7 percent further reduction in homocysteine concentrations, whereas vitamin B 6 had no significant additional effect (8). Quinlivan et al. (33) investigated the relation between both folate and vitamin B 12 status and plasma homocysteine concentrations in two groups of healthy subjects pre- and postsupplementation with folic acid. They observed that, after supplementation, the usual dependency of homocysteine on folate diminished, and vitamin B 12 became the main determinant of homocysteine concentrations (33). Homocysteine is formed from methionine. Thus, increased intake of methionine could lead to an increase in blood homocysteine and consequently to an increased risk of stroke. In this study, we observed no significant association between methionine intake and stroke. Our study has certain strengths and limitations that deserve mention. An advantage of this study is the large number of stroke cases, especially cerebral infarctions, which provided ample statistical power to detect associations. Furthermore, because no national folic acid fortification of cereal grain products has been initiated in Finland, folate

7 960 Larsson et al. fortification cannot have affected our results. The detailed data on multiple cardiovascular risk factors allowed adjustment for potential confounders, but there is still the possibility of residual or unmeasured confounding. Moreover, because of the observational design of our study, we cannot rule out the possibility that it may not be folate that is etiologically important but something else in high folate foods. Misclassification of dietary intake could have led to an underestimation of the associations of B vitamin and methionine intakes with risk of stroke in this study. Dietary intake was assessed at baseline only, which may have contributed to misclassification because of dietary changes during follow-up. Finally, our results may not be generalizable to nonsmokers, particularly as smokers have a higher risk of stroke and tend to have marginal folate status (34). In summary, our results in men suggest that a high dietary folate intake may reduce the risk of cerebral infarction, particularly among those with a high vitamin B 12 intake and among those with no history of coronary heart disease. Although these observational data do not prove a causal relation, they indicate that high consumption of folate-rich foods (e.g., whole grains, green leafy vegetables, oranges, and legumes) may play a role in the prevention of stroke. ACKNOWLEDGMENTS The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study was supported by Public Health Service contracts N01-CN-45165, N01-RC-45035, and N01-RC from the US National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland. Susanna C. Larsson s research at the National Public Health Institute in Helsinki, Finland, was supported by a grant from the Swedish Council for Working Life and Social Research. Conflict of interest: none declared. REFERENCES 1. 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