The Journal of Nutrition. First published ahead of print June 10, 2015 as doi: 10.3945/jn.114.209296. The Journal of Nutrition Nutrition and Disease
Choline and Betaine Intakes Are Not Associated with Cardiovascular Disease Mortality in Japanese Men and Women1–3 Chisato Nagata,4* Keiko Wada,4 Takashi Tamura,4 Kie Konishi,4 Toshiaki Kawachi,4 Michiko Tsuji,4,5 and Kozue Nakamura4,6 4 Department of Epidemiology and Preventive Medicine, Gifu University Graduate School of Medicine, Gifu, Japan; 5Department of Food Science and Nutrition, Nagoya Women’s University, Nagoya, Japan; and 6Department of Food and Nutrition, Gifu City WomenÕs College, Gifu, Japan
Background: Dietary intakes of betaine and choline may reduce the risk of cardiovascular disease; however, epidemiologic evidence is limited. Seafood is a rich source of betaine and is a popular traditional food in Japan. Objective: We examined the associations of betaine and choline intakes with cardiovascular disease mortality in a population-based cohort study in Japan. Methods: Study subjects were 13,355 male and 15,724 female residents of Takayama City, Japan, who were aged $35 y and enrolled in 1992. Their diets were assessed by a validated food frequency questionnaire. Deaths from coronary heart disease and stroke were identified from death certificates over 16 y. Results: During follow-up, we documented 308 deaths from coronary heart disease and 676 deaths from stroke (393 from ischemic and 153 from hemorrhagic strokes). Compared with the lowest quartile, the second, third, and highest quartiles of betaine intake were significantly associated with a decreased risk of mortality from coronary heart disease in men after controlling for covariates. The HRs were 0.58 (95% CI: 0.36, 0.93), 0.62 (95% CI: 0.39, 0.998), and 0.60 (95% CI: 0.37, 0.97), respectively. The trend was not statistically significant (P = 0.08). There was no significant association between betaine intake and the risk of mortality from ischemic stroke. In women, betaine intake was unrelated to mortality from coronary heart disease and stroke (P = 0.32 and 0.73, respectively, for interaction by sex). There was no significant association between choline intake and cardiovascular disease mortality in men or women. Conclusion: Overall, we found no clear evidence of significant associations between choline and betaine intakes and cardiovascular disease mortality in Japanese men and women. J Nutr doi: 10.3945/jn.114.209296.
Keywords:
betaine, choline, cohort studies, coronary heart disease, seafood, stroke
Introduction Choline and betaine promote homocysteine remethylation to Met, and, thereby, intakes of choline and betaine, as methyl donors, could affect homocysteine concentrations (1). High intakes of betaine and choline have been associated with lower homocysteine concentrations (2, 3). Intervention studies have shown that choline and betaine supplementations lowered fasting homocysteine or post–Met-load homocysteine concentrations (4, 5). Some studies have also shown that dietary
1 Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 2 Author disclosures: C Nagata, K Wada, T Tamura, K Konishi, T Kawachi, M Tsuji, and K Nakamura, no conflicts of interest. 3 Supplemental Tables 1–4 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. * To whom correspondence should be addressed. E-mail: [email protected].
intakes of betaine and choline were inversely associated with inflammatory markers, such as C-reactive protein, IL-6, and TNF-a [reviewed by Rajaie and Esmaillzadeh (6)]. High concentrations of homocysteine are associated with an increased risk of cardiovascular disease, such as stroke and coronary heart disease (7, 8). Inflammation plays a role in atherogenesis (9). Therefore, there is a hypothesis that intakes of betaine and choline may protect against cardiovascular disease (1). On the other hand, the metabolic pathway of dietary intakes of betaine and choline involving intestinal microbiota has been implicated in the pathogenesis of cardiovascular disease (10). So far, to our knowledge, 2 studies prospectively examined the association between intakes of betaine and choline and cardiovascular disease (11, 12), and both of them observed no association. We examined whether intakes of betaine and choline were associated with the risk of mortality from coronary heart disease and stroke in a Japanese cohort (Takayama study). Seafood is rich in
ã 2015 American Society for Nutrition. Manuscript received December 22, 2014. Initial review completed January 26, 2015. Revision accepted May 21, 2015. doi: 10.3945/jn.114.209296.
Copyright (C) 2015 by the American Society for Nutrition
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Abstract
betaine (13), and a high intake of seafood is one characteristic of the Japanese diet. It is of interest to examine this association in a Japanese population.
Methods Study population. The Takayama study is a prospective cohort study with the primary objective of investigating associations of diet and other lifestyle factors with cancer and other chronic diseases (14). A total of 31,552 residents of Takayama City, Gifu, aged $35 y, were enrolled in the study in 1992. The participation rate was 85.3%. The present study included 29,079 subjects with no history of cancer, stroke, or coronary heart disease. The details of the study design and subjects have been described previously (14, 15).
Follow-up and endpoints. The study endpoint was deaths from coronary heart disease and stroke that occurred between the baseline (1 September 1992) and 1 October 2008. Deaths and their causes occurring in this cohort were identified from death certificates provided by the Legal Affairs Bureau. The causes of deaths were coded according to the International Classification of Diseases (ICD). We included deaths from coronary heart disease (ICD-9 codes 410–414 and ICD-10 codes I20– I25) and stroke (ICD-9 codes 430–448 and ICD-10 codes I60–I69). Stroke was further classified as subarachnoid hemorrhage (ICD-9 code 430 and ICD-10 codes I60 and I69.0), intracerebral hemorrhage (ICD-9 code 431 and ICD-10 codes I61 and I69.1), ischemic strokes (ICD-9 code 434 and ICD-10 codes I63 and I69.3), and stroke of undetermined type. Information concerning subjects who moved away from Takayama City during the course of the study was obtained from the residential registers or family registers. During the study period, 1912 persons (6.1%) moved out of Takayama City. The date of emigration was unknown for 104 subjects (0.8%). They were censored at the latest date when they were known to reside in the city. This study was approved by the Ethical Board of the Gifu University Graduate School of Medicine. 2 of 6
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Results Baseline characteristics of the study population according to sex and quartile of energy-adjusted betaine and choline intakes are presented in Table 1. High intakes of betaine and choline were associated with more years of education, lower total energy, and higher intakes of PUFAs, fruits, folate, vitamin B-6, vitamin B-12, and Met. Major food groups supplying betaine were seafood, cereals/ potatoes/starches, and vegetables. The contributions (as a percentage of total amount of betaine) of these food groups were 60.4%, 20.6%, and 6.1%, respectively, in men and 58.6%,
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Baseline data collection. Diet including alcohol intake was assessed with a 169-item semiquantitative FFQ at the baseline. Details of the method of calculating nutrient intakes from the FFQ and its reliability and validity were previously described (15, 16). Nutrient values in foods including folate and B vitamins were obtained from the Japanese Standard Tables of Food Composition, 5th revised and enlarged edition, published by the Science and Technology Agency of Japan (17). FA composition was determined using data published by Sasaki et al. (18). Betaine and choline content data are not included in the Japanese Standard Tables of Food Composition. Studies reporting the values of betaine or choline from analyses of Japanese foods, except for seafood, are scarce. Therefore, betaine and choline intakes were estimated using the USDA database (19, 20). When data were not available, values published by de Zwart et al. (21), Zeizel et al. (22), and Lewis et al. (23) (choline only) were used. Previous studies on dietary betaine and choline conducted among non-US populations have used these data (12, 24–26). Total choline is the sum of free choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, and sphingomyelin. For some foods, such as taro, lotus root, and shellfish, including prawn, scallop, squid, octopus, etc., that are not available in the USDA data set or aforementioned articles (21, 22), the betaine values reported by Japanese researchers were used (27–36). The validity of the values for betaine and choline intakes could not be directly tested. However, as in another study on betaine and choline intakes and cardiovascular disease, we examined correlations between the questionnaire and 12 daily diet records kept over a 1-y period for the major food sources of betaine and choline; the Spearman correlation coefficients were 0.20 in men and 0.49 in women for cereals/potatoes/starches, 0.61 in men and 0.33 in women for seafood, and 0.23 in men and 0.51 in women for eggs. Those for folate, vitamin B-12, and Met were 0.54, 0.73, and 0.34, respectively, in men, and 0.46, 0.24, and 0.64, respectively, in women. Information about demographic characteristics, smoking, physical activity, and medical and reproductive histories were obtained by a self-administered questionnaire. The details are described elsewhere (15, 37).
Statistical analyses. We calculated person-years of follow-up for each subject from the date of response to baseline questionnaire to the date of death, the date of emigration out of Takayama, or the end of the follow-up (1 October 2008), whichever came first. All nutrients were energy adjusted by the residual method proposed by Willett (38). Study subjects were divided into quartiles according to their dietary intakes of betaine and choline. The characteristics of the study subjects according to betaine and choline intakes were assessed by the linear regression analysis for continuous variables and the Cochran-Armitage test for categorical variables. The HRs and their 95% CIs for mortality from coronary heart disease, total stroke, ischemic stroke, and hemorrhagic (subarachnoid hemorrhage and cerebral hemorrhage) stroke for each category were calculated using the Cox proportional hazard model. The lowest quartile was used as the reference. Tests for trend were performed by assessing the median value to each quartile and treating this variable as a continuous variable. In multivariate models, we adjusted for nondietary factors including age, marital status (married or not married), level of education (#11 y, 12–14 y, or $15 y), BMI (in quintile), smoking status (never, former, current with #30 y of smoking, or current and >30 y of smoking for men and never, former, or current for women), physical activity score, alcohol consumption (in quartile for men and categorized as those who drank no alcohol, equal to or less than median, or more than the median for women), use of vitamin supplements and aspirin (yes or no), histories of diabetes and hypertension (yes or no), menopausal status (premenopausal or postmenopausal; women only), and dietary factors including intakes of SFAs, PUFAs, salt, folate, and fruits. Similarly, we estimated the HRs of cardiovascular disease mortality for intake of the individual choline-containing compounds. The USDA database (Release 2, 2008) (19) showed that betaine concentrations were generally lower than those previously reported (Release 1, 2004) (39). Shrimp (mixed species, canned) is the only shellfish for which betaine concentration was reported in both releases. The betaine concentration of shrimp was corrected from 33 mg/100 g in Release 1 (2004) (39) to 23 mg/100 g in Release 2 (2008) (19). Thus, there might be a concern that the values reported by Japanese studies before 2008 may be also overestimates. Therefore, in a sensitivity analysis, we repeated our analysis after multiplying the values obtained from the Japanese studies by 0.697 (23 mg /33 mg). Besides the USDA database and Japanese studies, de Zwart et al. (21) reported a betaine concentration for shellfish but for clams only (250 mg/100 g). We also conducted a sensitivity analysis by reallocating the USDA betaine values for shrimp (mixed species, canned) and crab (blue canned) to any kinds of shrimp and any kinds of crabs, respectively. For any other shellfish, the betaine concentration for clams reported by de Zwart et al. (21) was multiplied by 0.697 and then reallocated. B vitamins are cofactors in homocysteine metabolism (1). Because the association between betaine and choline intakes and cardiovascular disease may be dependent on folate, vitamin B-6, vitamin B-12, and Met intakes, we conducted subgroup analyses according to low and high intakes of these nutrients. The significance of interaction was tested by including a cross-product term of betaine or choline intakes (as a continuous variable) and folate, vitamin B-6, vitamin B-12, or Met intakes (as a dichotomous variable). All of the statistical analyses were performed using SAS programs. Significance was defined as 2-sided P < 0.05.
959 8.9 8.2 283 128 7.1 277 0.9 9.3 1070
,0.0001 0.0002 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
0.17 0.08 0.59 0.46 ,0.0001 0.0008 ,0.0001 ,0.0001
,0.0001
0.42 0.17
P-trend
Men
2800 6 15.5 6 13.4 6 298 6 127 6 12.3 6 393 6 1.8 6 8.5 6 1910 6 840 9.0 6.2 200 168 5.4 188 0.7 4.8 717
14.4 23.5 62.1 25.1 4.3 18.1 4.0 22.5 6 2.7 28.6 6 43.9 52.8 6 52.8
59.6 31.0 9.4
362 52.1 6 11.1 90.2
Q1, n = 3339
2700 20.3 19.2 495 135 17.6 607 2.2 14.6 2610
6 985 6 9.7 6 8.6 6 334 6 139 6 7.4 6 328 6 1.0 6 9.3 6 1110
19.1 32.4 48.5 30.0 4.3 19.9 8.6 22.5 6 2.8 26.5 6 41.0 37.6 6 35.1
57.3 29.6 13.1
614 55.9 6 12.6 92.0
O4, n = 3338
Choline
,0.0001 ,0.0001 ,0.0001 ,0.0001 0.005 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 ,0.0001 0.89 0.03 ,0.0001 0.50 0.02 ,0.0001
,0.0001
,0.0001 0.02
P-trend
2480 18.0 15.3 433 163 13.8 493 1.7 7.5 1970
6 840 6 10.5 6 7.5 6 315 6 180 6 6.5 6 286 6 0.8 6 4.3 6 820
83.8 3.7 12.5 35.4 7.0 16.2 2.1 22.0 6 2.9 19.4 6 31.6 9.0 6 20.9 60.5
69.7 26.8 3.5
170 55.2 6 12.9 75.2
Q1, n = 3931
2270 17.4 16.7 457 145 15.3 532 1.8 12.4 2190
6 811 6 8.2 6 7.4 6 281 6 126 6 6.3 6 260 6 0.7 6 7.3 6 909
80.3 4.6 15.1 36.0 7.4 17.6 2.9 22.2 6 2.9 19.3 6 28.8 9.1 6 17.2 56.0
62.2 31.7 6.1
377 53.9 6 12.0 78.6
Q4, n = 3931
Betaine
,0.0001 0.46 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 0.58 0.56 0.21 0.03 0.01 0.75 0.38 ,0.0001
,0.0001
,0.0001 ,0.0001
P-trend
2380 15.2 13.3 338 140 12.0 406 1.5 7.1 1760
6 783 6 8.8 6 6.4 6 217 6 166 6 5.4 6 192 6 0.6 6 4.0 6 696
79.2 4.5 16.3 32.8 7.6 16.0 1.7 22.0 6 3.0 18.9 6 30.7 9.9 6 22.0 53.9
68.7 26.9 4.4
307 53.7 6 12.7 76.1
Q1, n = 3931
Women
2280 6 19.0 6 17.7 6 531 6 155 6 16.3 6 602 6 1.9 6 11.7 6 2300 6
807 8.8 7.5 331 137 6.6 304 0.8 7.1 923
84.8 4.0 11.2 38.9 6.5 18.6 3.9 22.1 6 3.0 19.9 6 29.4 7.5 6 15.2 63.2
64.9 29.8 5.3
525 56.0 6 12.7 74.7
Q4, n = 3931
Choline
0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 ,0.0001 0.03 ,0.007 ,0.0001 0.33 0.31 ,0.0001 ,0.0001
0.01
,0.0001 0.30
P-trend
1
Values are means 6 SDs or %. P values are based on linear regression analysis for continuous variables and on the Cochran-Armitage test for categorical variables. Numbers of missing data: marital status, 129 men and 255 women; years of education, 161 men and 247 women; smoking status, 386 men and 1634 women; BMI, 744 men and 897 women; aspirin use, 68 men and 101 women, and use of vitamin supplements, 785 men and 985 women. MET, metabolic equivalent; Q1, quartile 1; Q4, quartile 4.
2730 6 18.9 6 18.1 6 432 6 129 6 16.7 6 546 6 2.1 6 15.6 6 2530 6
16.9 28.8 54.3 28.3 4.6 19.8 7.9 22.8 6 2.9 24.9 6 39.4 39.6 6 36.8
16.2 27.2 56.6 26.2 4.4 19.2 4.7 22.6 6 2.7 31.8 6 45.1 55.5 6 51.5
6 891 6 10.8 6 7.6 6 294 6 185 6 6.7 6 285 6 0.8 6 5.2 6 902
50.4 34.0 15.6
64.3 27.0 8.7
2970 18.6 16.0 393 147 14.5 488 2.0 9.3 2200
463 53.5 6 11.9 92.1
209 53.2 6 11.6 90.9
Median, mg/d Age, y Married, % Years of education, % #11 12–14 $15 Smoking, % Never Former Current Use of vitamin supplements, % Use of aspirin, % History of hypertension, % History of diabetes, % BMI, kg/m2 Exercise, METs h21 wk21 Alcohol intake, mg/d Postmenopausal, % Dietary intake Total energy, kcal/d SFAs, g/d PUFAs, g/d Vegetables, g/d Fruits, g/d Salts, g/d Folate, μg/d Vitamin B-6, mg/d Vitamin B-12, μg/d Met, mg/d
Q4, n = 3338
Q1, n = 3339
Betaine
Baseline characteristics of Japanese men and women in the lowest and highest quartiles of betaine and choline intakes (n = 29,079)1
Basic characteristics
TABLE 1
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Betaine and choline and cardiovascular mortality
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24.0%, and 7.6%, respectively, in women. For choline intake, the major food groups were eggs (25.5%), seafood (15.2%), and meats (14.8%) in men and eggs (25.9%), seafood (13.9%), and soy foods (13.0%) in women. During the 16 y of follow-up, there were 308 coronary heart disease deaths and 676 stroke deaths. In men, the second, third, and highest quartiles of betaine intake were significantly associated with decreased risk of mortality from coronary heart disease compared with the lowest quartile of intake; the HRs were 0.58 (P = 0.02), 0.62 (P = 0.049), and 0.60 (P = 0.04), respectively. The linear trend was not statistically significant (P = 0.08; Table 2). The second quartile of betaine intake was significantly associated with a decreased risk of mortality from ischemic stroke (HR = 0.59, P = 0.02). However, the HR for the highest quartile of betaine intake was not statistically significant
(HR = 0.67, P = 0.09). There was no significant association between betaine intake and the risk of mortality from coronary heart disease and stroke in women (P values for interaction by sex were 0.32 and 0.73, respectively). Choline intake was not associated with the risk of mortality from coronary heart disease or stroke in men or women. The results from detailed analysis regarding individual cholinecontaining compounds are shown in Supplemental Table 1. There were no significant associations except that choline from sphingomyelin was positively associated with the risk of mortality from coronary heart disease in men and that choline from phosphocholine was inversely associated with the risk of mortality from hemorrhagic stroke in women. To examine if the observed associations may be caused by some other component of foods containing high betaine or
Men
Betaine Median, mg/d Person-years Coronary heart disease Cases, n Age-adjusted HR Multivariate HR1 Total stroke Cases, n Age-adjusted HR Multivariate HR1 Ischemic stroke Cases, n Age-adjusted HR Multivariate HR1 Hemorrhagic stroke Cases, n Age-adjusted HR Multivariate HR1 Choline Median, mg/d Person-years Coronary heart disease Cases, n Age-adjusted HR Multivariate HR1 Total stroke Cases, n Age-adjusted HR Multivariate HR1 Ischemic stroke Cases, n Age-adjusted HR Multivariate HR1 Hemorrhagic stroke Cases, n Age-adjusted HR Multivariate HR1
Women
Q1, n = 3339
Q2, n = 3339
Q3, n = 3339
Q4, n = 3338
208 47,092
287 46,079
350 44,644
463 45,484
Q1, P-trend n = 3931
170 57,139
Q2, n = 3931
Q3, n = 3931
Q4, n = 3931
239 55,789
288 56,456
377 57,699
P-trend
46 1.0 1.0
32 39 37 0.64 (0.41, 1.01) 0.74 (0.48, 1.14) 0.79 (0.51, 1.21) 0.58 (0.36, 0.93) 0.62 (0.39, 0.998) 0.60 (0.37, 0.97)
0.41 0.08
37 1.0 1.0
44 43 30 1.05 (0.68, 1.63) 1.21 (0.77, 1.87) 0.99 (0.61, 1.61) 0.94 (0.58, 1.50) 0.98 (0.61, 1.58) 0.90 (0.53, 1.51)
0.90 0.74
76 1.0 1.0
74 0.87 (0.63, 1.19) 0.79 (0.56, 1.10)
99 1.07 (0.79, 1.45) 0.90 (0.65, 1.27)
79 0.98 (0.72, 1.34) 0.84 (0.59, 1.20)
0.81 0.54
76 1.0 1.0
125 86 62 1.39 (1.04, 1.85) 1.17 (0.86, 1.59) 1.06 (0.75, 1.48) 1.32 (0.97, 1.79) 1.06 (0.76, 1.48) 1.04 (0.72, 1.49)
0.99 0.78
47 1.0 1.0
38 0.67 (0.44, 1.03) 0.59 (0.37, 0.92)
62 0.98 (0.67, 1.43) 0.75 (0.49, 1.17)
46 0.85 (0.57, 1.28) 0.67 (0.42, 1.07)
0.83 0.22
43 1.0 1.0
74 47 36 1.38 (0.95, 2.01) 1.12 (0.74, 1.70) 1.15 (0.74, 1.79) 1.39 (0.93, 2.10) 1.02 (0.66, 1.60) 1.15 (0.71, 1.85)
0.76 0.94
24 1.0 1.0
34 1.39 (0.82, 2.34) 1.32 (0.77, 2.27)
32 1.30 (0.76, 2.21) 1.22 (0.69, 2.16)
30 1.27 (0.75, 2.18) 1.23 (0.68, 2.22)
0.50 0.66
30 1.0 1.0
39 24 29 1.44 (0.89, 2.31) 1.07 (0.64, 1.80) 0.87 (0.49, 1.53) 1.28 (0.77, 2.14) 0.93 (0.53, 1.63) 0.79 (0.43, 1.46)
0.43 0.27
362 47,479
445 46,460
513 45,498
614 43,863
31 1.0 1.0
33 1.04 (0.77, 1.41) 0.95 (0.56, 1.61)
41 1.22 (0.91, 1.64) 0.95 (0.54, 1.65)
49 1.15 (0.84, 1.57) 1.08 (0.57, 2.04)
0.25 0.74
30 1.0 1.0
48 40 36 1.31 (0.83, 2.07) 1.15 (0.72, 1.85) 1.05 (0.65, 1.71) 1.08 (0.64, 1.84) 0.91 (0.51, 1.64) 0.80 (0.40, 1.60)
0.97 0.39
59 1.0 1.0
75 1.07 (0.76, 1.50) 1.01 (0.70, 1.46)
93 1.11 (0.80, 1.54) 1.00 (0.67, 1.48)
101 1.14 (0.82, 1.57) 0.98 (0.62, 1.55)
0.44 0.92
74 1.0 1.0
108 81 86 1.13 (0.84, 1.52) 0.90 (0.66, 1.24) 1.01 (0.74, 1.38) 1.06 (0.75, 1.50) 0.85 (0.57, 1.25) 1.02 (0.65, 1.60)
0.73 0.84
32 1.0 1.0
37 0.88 (0.55, 1.42) 0.78 (0.47, 1.31)
61 1.13 (0.73, 1.74) 0.88 (0.52, 1.49)
63 1.07 (0.70, 1.65) 0.80 (0.43, 1.46)
0.51 0.61
38 1.0 1.0
61 50 51 1.16 (0.78, 1.75) 1.03 (0.68, 1.57) 1.16 (0.77, 1.77) 1.17 (0.73, 1.87) 1.04 (0.62, 1.74) 1.17 (0.64, 2.14)
0.61 0.76
27 1.0 1.0
32 1.13 (0.68, 1.88) 1.18 (0.67, 2.05)
26 0.87 (0.51, 1.50) 0.92 (0.49, 1.74)
35 1.16 (0.70, 1.92) 1.28 (0.63, 2.63)
0.73 0.60
28 1.0 1.0
44 29 21 1.11 (0.69, 1.79) 0.72 (0.42, 1.22) 0.86 (0.52, 1.43) 0.94 (0.53, 1.65) 0.60 (0.31, 1.16) 0.76 (0.36, 1.60)
0.32 0.34
307 57,548
388 56,478
442 56,721
525 56,305
1 Adjusted for age, energy, BMI, physical activity, smoking status, education, marital status, use of vitamin supplements and aspirin, and histories of diabetes and hypertension, and intakes of alcohol, SFAs, PUFAs, folate, salt, and fruits. For women, additionally adjusted for menopausal status.
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TABLE 2 HRs with 95% CIs for cardiovascular disease mortality by quartiles of betaine and choline intakes in Japanese men and women (1992–2008)
choline, further adjustments for major sources of betaine and choline intakes were made (Supplemental Tables 2 and 3). The results were not altered substantially. The sensitivity analyses revealed that the different methods estimating betaine intake did not affect the results substantially (Supplemental Table 4). The association between betaine intake and mortality from coronary heart disease as well as ischemic stroke in men did not differ significantly according to low and high intakes of folate, vitamin B-6, vitamin B-12, and Met (Supplemental Table 4). Exclusion of deaths during the first 4 y also did not alter the results substantially (Supplemental Table 4).
Discussion
Acknowledgments We thank Shougen Matsushita and Takehiko Minakuchi for their help in data collection. CN organized the study and wrote the manuscript; KW, TT, KK, TK, and MT conducted the data analysis and interpreted the data; KN helped supervise the field activities and interpret the data. All authors read and approved the final manuscript.
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Our study, as well as previous studies, observed no association between choline intake and cardiovascular disease (11, 12). The major fate of dietary choline is conversion to phosphatidylcholine (1). Phosphatidylcholine is essential for the secretion of VLDL from the liver (40). The TG-raising effect of phosphatidylcholine is suggested, which may contribute to negation of the inverse association with the risk of cardiovascular disease (41). Although intestinal microbial metabolism of dietary phosphatidylcholine has been implicated in the pathogenesis of atherosclerosis (10, 42), intake of choline from phosphatidylcholine was unrelated to mortality from coronary heart disease as well as stroke in our study. Because the bioavailability of different choline sources might differ, further studies on the association of intake of individual choline compounds with the risk of cardiovascular disease are warranted. Although our results were generally null, previous studies suggested that betaine could potentially influence cardiovascular disease risk. Several studies have found betaine to be a determinant for homocysteine concentration (2, 3, 6). Metaanalyses have shown that high homocysteine concentration is associated with the risk of coronary heart disease (7) and ischemic stroke (8). Although it is still unclear whether these relations are causal (43), it has been postulated that homocysteine may increase abnormal platelet activity and blood coagulation, which lead to thrombotic disorders (44). A high betaine intake may increase betaine availability and enhance remethylation of homocysteine (back to Met). Another study conducted among US men and women observed no association of betaine intake with the risk of coronary heart disease (11). The major source of dietary betaine is cereal foods in Western populations but is seafood in our population (45). Some studies have reported that high intake of seafood was associated with a decreased risk of coronary heart disease or ischemic stroke (46). Long-chain (n–3) PUFAs, which are mainly derived from fish and shellfish, are thought to be cardioprotective, antiarrhythmic, and antithrombotic (47). Although adjustment for seafood did not alter the present results, we cannot deny the possibility that long-chain (n–3) PUFAs or other components in fish or shellfish, rather than betaine, may be relevant to the risk of cardiovascular disease. On the other hand, the present study would suggest the possibility that betaine in seafood may be driving the association of seafood intake with cardiovascular disease. The strengths of the present study included its prospective design, good participation rate, representation of the general population, long follow-up, and information on potential confounders. Several limitations should be considered. It is known that seafood, especially marine invertebrates (10), is rich
in betaine. However, betaine data for invertebrates are scarce, probably because of the low consumption of shellfish in Western populations. Although adjustment for seafood intake did not alter the results and the sensitivity analyses assigning the different concentration values to shellfish did not alter the results substantially, measurement error could lead to biased estimates. Expansion of the reference database to include more seafood items is necessary. The sample size was limited, which precluded analyses on subtypes of stroke with small numbers of deaths. The use of mortality instead of incidence data prevented us from distinguishing the effect of diet on incidence, survival, or both. Our questionnaire was designed to measure an individualÕs relative intakes of nutrients rather than absolute values. Although we presented the mean values for nutrients in the table, some of them may be overestimated by our questionnaire. The mean values estimated from the FFQ were generally higher than those estimated from 12 daily diet records; e.g., the mean estimate of folate in the former was 19.7% higher in men and 14.8% higher in women than in the latter. Dietary habits measured only at baseline and the single assessment of dietary intake may not be reflective of long-term dietary exposure. Therefore, misclassification of betaine and choline intakes may have occurred because of measurement error and change of diet over time. The influence of underlying diseases or preclinical signs on dietary intake was possible, especially in the use of mortality data. In addition, we could not assess the very recent diet, which may have great impact on cardiovascular disease mortality with a very short induction time. Although numerous lifestyle and dietary factors were adjusted in the analyses, residual confounders or unmeasured variables could account for the observed associations. In conclusion, we found no clear evidence of significant associations between choline and betaine intakes and cardiovascular disease mortality in Japanese men and women. Our findings from subgroup analysis concerning betaine intake and mortality from coronary heart disease in men need confirmation in future studies because we cannot rule out that these findings could be caused by bias or chance.
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Rajaie S, Esmaillzadeh A. Dietary choline and betaine intakes and risk of cardiovascular diseases: review of epidemiologic evidence. ARYA Atheroscler 2011;7:78–86. Humphrey LL, Fu R, Rogers K, Freeman M, Helfand M. Homocysteine level and coronary heart disease incidence: a systematic review and meta-analysis. Mayo Clin Proc 2008;83:1203–12. He Y, Li Y, Chen Y, Feng L, Nie Z. Homocysteine level and risk of different stroke types: a meta-analysis of prospective observational studies. Nutr Metab Cardiovasc Dis 2014;24:1158–65. Golia E, Limongelli G, Natale F, Fimiani F, Maddaloni V, Pariggiano I, Bianchi R, Crisci M, D’Acierno L, Giordano R, et al. Inflammation and cardiovascular disease: from pathogenesis to therapeutic target. Curr Atheroscler Rep 2014;16:435. Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011;472:57–63. Bidulescu A, Chambless LE, Siega-Riz AM, Zeisel SH, Heiss G. Usual choline and betaine dietary intake and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study. BMC Cardiovasc Disord 2007;7:20. Dalmeijer GW, Olthof MR, Verhoef P, Bots ML, van der Schouw YT. Prospective study on dietary intakes of folate, betaine, and choline and cardiovascular disease risk in women. Eur J Clin Nutr 2008;62:386–94. Craig SA. Betaine in human nutrition. Am J Clin Nutr 2004;80:539–49. Shimizu H. The Basic Report on Takayama Study. Gifu (Japan): Department of Public Health, Gifu University School of Medicine; 1996. Nagata C, Wada K, Tamura T, Kawachi T, Konishi K, Tsuji M, Nakamura K. Dietary intakes of glutamic acid and glycine are associated with stroke mortality in Japanese adults. J Nutr 2015;145:720–8. Shimizu H, Ohwaki A, Kurisu Y, Takatsuka N, Ido M, Kawakami N, Nagata C, Inaba S. Validity and reproducibility of a quantitative food frequency questionnaire for a cohort study in Japan. Jpn J Clin Oncol 1999;29:38–44. Council for Science and Technology; Ministry of Education, Culture, Sports, Science and Technology, Japan. Standard tables of food composition in Japan. 5th Revised and enlarged ed. Tokyo: Kagawa Education Institute of Nutrition; 2008. Sasaki S, Kobayashi M, Tsugane S. Development of substituted fatty acid composition table for the use in nutritional epidemiologic studies for Japanese populations: its methodological backgrounds and the evaluation. J Epidemiol 1999;9:190–207. USDA, Agriculture Research Service. USDA nutrient database for standard reference. Release 27. Washington (DC): Agricultural Research Service; 2011. Patterson KY, Bhagwat SA, Williams JR, Howe JC, Holden JM. USDA database for the choline content of common foods, Release 2 [Internet]. 2008 [cited 2014 Nov 5]. Available from: http://naldc.nal.usda.gov/ download/47335/PDF de Zwart FJ, Slow S, Payne RJ, Lever M, George PM, Gerrard JA, Chambers ST. Glycine betaine and glycine betaine analogues in common foods. Food Chem 2003;83:197–204. Zeisel SH, Mar MH, Howe JC, Holden JM. Concentrations of cholinecontaining compounds and betaine in common foods. J Nutr 2003;133:1302–7. Lewis ED, Kosik SJ, Zhao YY, Jacobs RL, Curtis JM, Field CJ. Total choline and choline-containing moieties of commercially available pulses. Plant Foods Hum Nutr 2014;69:115–21. Bidulescu A, Chambless LE, Siega-Riz AM, Zeisel SH, Heiss G. Repeatability and measurement error in the assessment of choline and betaine dietary intake: the Atherosclerosis Risk in Communities (ARIC) study. Nutr J 2009;8:14. Zhang CX, Pan MX, Li B, Wang L, Mo XF, Chen YM, Lin FY, Ho SC. Choline and betaine intake is inversely associated with breast cancer risk: a two-stage case-control study in China. Cancer Sci 2013;104: 250–8.
Choline and Betaine Intakes Are Not Associated with Cardiovascular Disease Mortality in Japanese Men and Women1–3 Chisato Nagata,4* Keiko Wada,4 Takashi Tamura,4 Kie Konishi,4 Toshiaki Kawachi,4 Michiko Tsuji,4,5 and Kozue Nakamura4,6 4 Department of Epidemiology and Preventive Medicine, Gifu University Graduate School of Medicine, Gifu, Japan; 5Department of Food Science and Nutrition, Nagoya Women’s University, Nagoya, Japan; and 6Department of Food and Nutrition, Gifu City WomenÕs College, Gifu, Japan
Background: Dietary intakes of betaine and choline may reduce the risk of cardiovascular disease; however, epidemiologic evidence is limited. Seafood is a rich source of betaine and is a popular traditional food in Japan. Objective: We examined the associations of betaine and choline intakes with cardiovascular disease mortality in a population-based cohort study in Japan. Methods: Study subjects were 13,355 male and 15,724 female residents of Takayama City, Japan, who were aged $35 y and enrolled in 1992. Their diets were assessed by a validated food frequency questionnaire. Deaths from coronary heart disease and stroke were identified from death certificates over 16 y. Results: During follow-up, we documented 308 deaths from coronary heart disease and 676 deaths from stroke (393 from ischemic and 153 from hemorrhagic strokes). Compared with the lowest quartile, the second, third, and highest quartiles of betaine intake were significantly associated with a decreased risk of mortality from coronary heart disease in men after controlling for covariates. The HRs were 0.58 (95% CI: 0.36, 0.93), 0.62 (95% CI: 0.39, 0.998), and 0.60 (95% CI: 0.37, 0.97), respectively. The trend was not statistically significant (P = 0.08). There was no significant association between betaine intake and the risk of mortality from ischemic stroke. In women, betaine intake was unrelated to mortality from coronary heart disease and stroke (P = 0.32 and 0.73, respectively, for interaction by sex). There was no significant association between choline intake and cardiovascular disease mortality in men or women. Conclusion: Overall, we found no clear evidence of significant associations between choline and betaine intakes and cardiovascular disease mortality in Japanese men and women. J Nutr doi: 10.3945/jn.114.209296.
Keywords:
betaine, choline, cohort studies, coronary heart disease, seafood, stroke
Introduction Choline and betaine promote homocysteine remethylation to Met, and, thereby, intakes of choline and betaine, as methyl donors, could affect homocysteine concentrations (1). High intakes of betaine and choline have been associated with lower homocysteine concentrations (2, 3). Intervention studies have shown that choline and betaine supplementations lowered fasting homocysteine or post–Met-load homocysteine concentrations (4, 5). Some studies have also shown that dietary
1 Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 2 Author disclosures: C Nagata, K Wada, T Tamura, K Konishi, T Kawachi, M Tsuji, and K Nakamura, no conflicts of interest. 3 Supplemental Tables 1–4 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. * To whom correspondence should be addressed. E-mail: [email protected].
intakes of betaine and choline were inversely associated with inflammatory markers, such as C-reactive protein, IL-6, and TNF-a [reviewed by Rajaie and Esmaillzadeh (6)]. High concentrations of homocysteine are associated with an increased risk of cardiovascular disease, such as stroke and coronary heart disease (7, 8). Inflammation plays a role in atherogenesis (9). Therefore, there is a hypothesis that intakes of betaine and choline may protect against cardiovascular disease (1). On the other hand, the metabolic pathway of dietary intakes of betaine and choline involving intestinal microbiota has been implicated in the pathogenesis of cardiovascular disease (10). So far, to our knowledge, 2 studies prospectively examined the association between intakes of betaine and choline and cardiovascular disease (11, 12), and both of them observed no association. We examined whether intakes of betaine and choline were associated with the risk of mortality from coronary heart disease and stroke in a Japanese cohort (Takayama study). Seafood is rich in
ã 2015 American Society for Nutrition. Manuscript received December 22, 2014. Initial review completed January 26, 2015. Revision accepted May 21, 2015. doi: 10.3945/jn.114.209296.
Copyright (C) 2015 by the American Society for Nutrition
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Abstract
betaine (13), and a high intake of seafood is one characteristic of the Japanese diet. It is of interest to examine this association in a Japanese population.
Methods Study population. The Takayama study is a prospective cohort study with the primary objective of investigating associations of diet and other lifestyle factors with cancer and other chronic diseases (14). A total of 31,552 residents of Takayama City, Gifu, aged $35 y, were enrolled in the study in 1992. The participation rate was 85.3%. The present study included 29,079 subjects with no history of cancer, stroke, or coronary heart disease. The details of the study design and subjects have been described previously (14, 15).
Follow-up and endpoints. The study endpoint was deaths from coronary heart disease and stroke that occurred between the baseline (1 September 1992) and 1 October 2008. Deaths and their causes occurring in this cohort were identified from death certificates provided by the Legal Affairs Bureau. The causes of deaths were coded according to the International Classification of Diseases (ICD). We included deaths from coronary heart disease (ICD-9 codes 410–414 and ICD-10 codes I20– I25) and stroke (ICD-9 codes 430–448 and ICD-10 codes I60–I69). Stroke was further classified as subarachnoid hemorrhage (ICD-9 code 430 and ICD-10 codes I60 and I69.0), intracerebral hemorrhage (ICD-9 code 431 and ICD-10 codes I61 and I69.1), ischemic strokes (ICD-9 code 434 and ICD-10 codes I63 and I69.3), and stroke of undetermined type. Information concerning subjects who moved away from Takayama City during the course of the study was obtained from the residential registers or family registers. During the study period, 1912 persons (6.1%) moved out of Takayama City. The date of emigration was unknown for 104 subjects (0.8%). They were censored at the latest date when they were known to reside in the city. This study was approved by the Ethical Board of the Gifu University Graduate School of Medicine. 2 of 6
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Results Baseline characteristics of the study population according to sex and quartile of energy-adjusted betaine and choline intakes are presented in Table 1. High intakes of betaine and choline were associated with more years of education, lower total energy, and higher intakes of PUFAs, fruits, folate, vitamin B-6, vitamin B-12, and Met. Major food groups supplying betaine were seafood, cereals/ potatoes/starches, and vegetables. The contributions (as a percentage of total amount of betaine) of these food groups were 60.4%, 20.6%, and 6.1%, respectively, in men and 58.6%,
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Baseline data collection. Diet including alcohol intake was assessed with a 169-item semiquantitative FFQ at the baseline. Details of the method of calculating nutrient intakes from the FFQ and its reliability and validity were previously described (15, 16). Nutrient values in foods including folate and B vitamins were obtained from the Japanese Standard Tables of Food Composition, 5th revised and enlarged edition, published by the Science and Technology Agency of Japan (17). FA composition was determined using data published by Sasaki et al. (18). Betaine and choline content data are not included in the Japanese Standard Tables of Food Composition. Studies reporting the values of betaine or choline from analyses of Japanese foods, except for seafood, are scarce. Therefore, betaine and choline intakes were estimated using the USDA database (19, 20). When data were not available, values published by de Zwart et al. (21), Zeizel et al. (22), and Lewis et al. (23) (choline only) were used. Previous studies on dietary betaine and choline conducted among non-US populations have used these data (12, 24–26). Total choline is the sum of free choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, and sphingomyelin. For some foods, such as taro, lotus root, and shellfish, including prawn, scallop, squid, octopus, etc., that are not available in the USDA data set or aforementioned articles (21, 22), the betaine values reported by Japanese researchers were used (27–36). The validity of the values for betaine and choline intakes could not be directly tested. However, as in another study on betaine and choline intakes and cardiovascular disease, we examined correlations between the questionnaire and 12 daily diet records kept over a 1-y period for the major food sources of betaine and choline; the Spearman correlation coefficients were 0.20 in men and 0.49 in women for cereals/potatoes/starches, 0.61 in men and 0.33 in women for seafood, and 0.23 in men and 0.51 in women for eggs. Those for folate, vitamin B-12, and Met were 0.54, 0.73, and 0.34, respectively, in men, and 0.46, 0.24, and 0.64, respectively, in women. Information about demographic characteristics, smoking, physical activity, and medical and reproductive histories were obtained by a self-administered questionnaire. The details are described elsewhere (15, 37).
Statistical analyses. We calculated person-years of follow-up for each subject from the date of response to baseline questionnaire to the date of death, the date of emigration out of Takayama, or the end of the follow-up (1 October 2008), whichever came first. All nutrients were energy adjusted by the residual method proposed by Willett (38). Study subjects were divided into quartiles according to their dietary intakes of betaine and choline. The characteristics of the study subjects according to betaine and choline intakes were assessed by the linear regression analysis for continuous variables and the Cochran-Armitage test for categorical variables. The HRs and their 95% CIs for mortality from coronary heart disease, total stroke, ischemic stroke, and hemorrhagic (subarachnoid hemorrhage and cerebral hemorrhage) stroke for each category were calculated using the Cox proportional hazard model. The lowest quartile was used as the reference. Tests for trend were performed by assessing the median value to each quartile and treating this variable as a continuous variable. In multivariate models, we adjusted for nondietary factors including age, marital status (married or not married), level of education (#11 y, 12–14 y, or $15 y), BMI (in quintile), smoking status (never, former, current with #30 y of smoking, or current and >30 y of smoking for men and never, former, or current for women), physical activity score, alcohol consumption (in quartile for men and categorized as those who drank no alcohol, equal to or less than median, or more than the median for women), use of vitamin supplements and aspirin (yes or no), histories of diabetes and hypertension (yes or no), menopausal status (premenopausal or postmenopausal; women only), and dietary factors including intakes of SFAs, PUFAs, salt, folate, and fruits. Similarly, we estimated the HRs of cardiovascular disease mortality for intake of the individual choline-containing compounds. The USDA database (Release 2, 2008) (19) showed that betaine concentrations were generally lower than those previously reported (Release 1, 2004) (39). Shrimp (mixed species, canned) is the only shellfish for which betaine concentration was reported in both releases. The betaine concentration of shrimp was corrected from 33 mg/100 g in Release 1 (2004) (39) to 23 mg/100 g in Release 2 (2008) (19). Thus, there might be a concern that the values reported by Japanese studies before 2008 may be also overestimates. Therefore, in a sensitivity analysis, we repeated our analysis after multiplying the values obtained from the Japanese studies by 0.697 (23 mg /33 mg). Besides the USDA database and Japanese studies, de Zwart et al. (21) reported a betaine concentration for shellfish but for clams only (250 mg/100 g). We also conducted a sensitivity analysis by reallocating the USDA betaine values for shrimp (mixed species, canned) and crab (blue canned) to any kinds of shrimp and any kinds of crabs, respectively. For any other shellfish, the betaine concentration for clams reported by de Zwart et al. (21) was multiplied by 0.697 and then reallocated. B vitamins are cofactors in homocysteine metabolism (1). Because the association between betaine and choline intakes and cardiovascular disease may be dependent on folate, vitamin B-6, vitamin B-12, and Met intakes, we conducted subgroup analyses according to low and high intakes of these nutrients. The significance of interaction was tested by including a cross-product term of betaine or choline intakes (as a continuous variable) and folate, vitamin B-6, vitamin B-12, or Met intakes (as a dichotomous variable). All of the statistical analyses were performed using SAS programs. Significance was defined as 2-sided P < 0.05.
959 8.9 8.2 283 128 7.1 277 0.9 9.3 1070
,0.0001 0.0002 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
0.17 0.08 0.59 0.46 ,0.0001 0.0008 ,0.0001 ,0.0001
,0.0001
0.42 0.17
P-trend
Men
2800 6 15.5 6 13.4 6 298 6 127 6 12.3 6 393 6 1.8 6 8.5 6 1910 6 840 9.0 6.2 200 168 5.4 188 0.7 4.8 717
14.4 23.5 62.1 25.1 4.3 18.1 4.0 22.5 6 2.7 28.6 6 43.9 52.8 6 52.8
59.6 31.0 9.4
362 52.1 6 11.1 90.2
Q1, n = 3339
2700 20.3 19.2 495 135 17.6 607 2.2 14.6 2610
6 985 6 9.7 6 8.6 6 334 6 139 6 7.4 6 328 6 1.0 6 9.3 6 1110
19.1 32.4 48.5 30.0 4.3 19.9 8.6 22.5 6 2.8 26.5 6 41.0 37.6 6 35.1
57.3 29.6 13.1
614 55.9 6 12.6 92.0
O4, n = 3338
Choline
,0.0001 ,0.0001 ,0.0001 ,0.0001 0.005 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 ,0.0001 0.89 0.03 ,0.0001 0.50 0.02 ,0.0001
,0.0001
,0.0001 0.02
P-trend
2480 18.0 15.3 433 163 13.8 493 1.7 7.5 1970
6 840 6 10.5 6 7.5 6 315 6 180 6 6.5 6 286 6 0.8 6 4.3 6 820
83.8 3.7 12.5 35.4 7.0 16.2 2.1 22.0 6 2.9 19.4 6 31.6 9.0 6 20.9 60.5
69.7 26.8 3.5
170 55.2 6 12.9 75.2
Q1, n = 3931
2270 17.4 16.7 457 145 15.3 532 1.8 12.4 2190
6 811 6 8.2 6 7.4 6 281 6 126 6 6.3 6 260 6 0.7 6 7.3 6 909
80.3 4.6 15.1 36.0 7.4 17.6 2.9 22.2 6 2.9 19.3 6 28.8 9.1 6 17.2 56.0
62.2 31.7 6.1
377 53.9 6 12.0 78.6
Q4, n = 3931
Betaine
,0.0001 0.46 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 0.58 0.56 0.21 0.03 0.01 0.75 0.38 ,0.0001
,0.0001
,0.0001 ,0.0001
P-trend
2380 15.2 13.3 338 140 12.0 406 1.5 7.1 1760
6 783 6 8.8 6 6.4 6 217 6 166 6 5.4 6 192 6 0.6 6 4.0 6 696
79.2 4.5 16.3 32.8 7.6 16.0 1.7 22.0 6 3.0 18.9 6 30.7 9.9 6 22.0 53.9
68.7 26.9 4.4
307 53.7 6 12.7 76.1
Q1, n = 3931
Women
2280 6 19.0 6 17.7 6 531 6 155 6 16.3 6 602 6 1.9 6 11.7 6 2300 6
807 8.8 7.5 331 137 6.6 304 0.8 7.1 923
84.8 4.0 11.2 38.9 6.5 18.6 3.9 22.1 6 3.0 19.9 6 29.4 7.5 6 15.2 63.2
64.9 29.8 5.3
525 56.0 6 12.7 74.7
Q4, n = 3931
Choline
0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001
,0.0001 ,0.0001 0.03 ,0.007 ,0.0001 0.33 0.31 ,0.0001 ,0.0001
0.01
,0.0001 0.30
P-trend
1
Values are means 6 SDs or %. P values are based on linear regression analysis for continuous variables and on the Cochran-Armitage test for categorical variables. Numbers of missing data: marital status, 129 men and 255 women; years of education, 161 men and 247 women; smoking status, 386 men and 1634 women; BMI, 744 men and 897 women; aspirin use, 68 men and 101 women, and use of vitamin supplements, 785 men and 985 women. MET, metabolic equivalent; Q1, quartile 1; Q4, quartile 4.
2730 6 18.9 6 18.1 6 432 6 129 6 16.7 6 546 6 2.1 6 15.6 6 2530 6
16.9 28.8 54.3 28.3 4.6 19.8 7.9 22.8 6 2.9 24.9 6 39.4 39.6 6 36.8
16.2 27.2 56.6 26.2 4.4 19.2 4.7 22.6 6 2.7 31.8 6 45.1 55.5 6 51.5
6 891 6 10.8 6 7.6 6 294 6 185 6 6.7 6 285 6 0.8 6 5.2 6 902
50.4 34.0 15.6
64.3 27.0 8.7
2970 18.6 16.0 393 147 14.5 488 2.0 9.3 2200
463 53.5 6 11.9 92.1
209 53.2 6 11.6 90.9
Median, mg/d Age, y Married, % Years of education, % #11 12–14 $15 Smoking, % Never Former Current Use of vitamin supplements, % Use of aspirin, % History of hypertension, % History of diabetes, % BMI, kg/m2 Exercise, METs h21 wk21 Alcohol intake, mg/d Postmenopausal, % Dietary intake Total energy, kcal/d SFAs, g/d PUFAs, g/d Vegetables, g/d Fruits, g/d Salts, g/d Folate, μg/d Vitamin B-6, mg/d Vitamin B-12, μg/d Met, mg/d
Q4, n = 3338
Q1, n = 3339
Betaine
Baseline characteristics of Japanese men and women in the lowest and highest quartiles of betaine and choline intakes (n = 29,079)1
Basic characteristics
TABLE 1
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Betaine and choline and cardiovascular mortality
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24.0%, and 7.6%, respectively, in women. For choline intake, the major food groups were eggs (25.5%), seafood (15.2%), and meats (14.8%) in men and eggs (25.9%), seafood (13.9%), and soy foods (13.0%) in women. During the 16 y of follow-up, there were 308 coronary heart disease deaths and 676 stroke deaths. In men, the second, third, and highest quartiles of betaine intake were significantly associated with decreased risk of mortality from coronary heart disease compared with the lowest quartile of intake; the HRs were 0.58 (P = 0.02), 0.62 (P = 0.049), and 0.60 (P = 0.04), respectively. The linear trend was not statistically significant (P = 0.08; Table 2). The second quartile of betaine intake was significantly associated with a decreased risk of mortality from ischemic stroke (HR = 0.59, P = 0.02). However, the HR for the highest quartile of betaine intake was not statistically significant
(HR = 0.67, P = 0.09). There was no significant association between betaine intake and the risk of mortality from coronary heart disease and stroke in women (P values for interaction by sex were 0.32 and 0.73, respectively). Choline intake was not associated with the risk of mortality from coronary heart disease or stroke in men or women. The results from detailed analysis regarding individual cholinecontaining compounds are shown in Supplemental Table 1. There were no significant associations except that choline from sphingomyelin was positively associated with the risk of mortality from coronary heart disease in men and that choline from phosphocholine was inversely associated with the risk of mortality from hemorrhagic stroke in women. To examine if the observed associations may be caused by some other component of foods containing high betaine or
Men
Betaine Median, mg/d Person-years Coronary heart disease Cases, n Age-adjusted HR Multivariate HR1 Total stroke Cases, n Age-adjusted HR Multivariate HR1 Ischemic stroke Cases, n Age-adjusted HR Multivariate HR1 Hemorrhagic stroke Cases, n Age-adjusted HR Multivariate HR1 Choline Median, mg/d Person-years Coronary heart disease Cases, n Age-adjusted HR Multivariate HR1 Total stroke Cases, n Age-adjusted HR Multivariate HR1 Ischemic stroke Cases, n Age-adjusted HR Multivariate HR1 Hemorrhagic stroke Cases, n Age-adjusted HR Multivariate HR1
Women
Q1, n = 3339
Q2, n = 3339
Q3, n = 3339
Q4, n = 3338
208 47,092
287 46,079
350 44,644
463 45,484
Q1, P-trend n = 3931
170 57,139
Q2, n = 3931
Q3, n = 3931
Q4, n = 3931
239 55,789
288 56,456
377 57,699
P-trend
46 1.0 1.0
32 39 37 0.64 (0.41, 1.01) 0.74 (0.48, 1.14) 0.79 (0.51, 1.21) 0.58 (0.36, 0.93) 0.62 (0.39, 0.998) 0.60 (0.37, 0.97)
0.41 0.08
37 1.0 1.0
44 43 30 1.05 (0.68, 1.63) 1.21 (0.77, 1.87) 0.99 (0.61, 1.61) 0.94 (0.58, 1.50) 0.98 (0.61, 1.58) 0.90 (0.53, 1.51)
0.90 0.74
76 1.0 1.0
74 0.87 (0.63, 1.19) 0.79 (0.56, 1.10)
99 1.07 (0.79, 1.45) 0.90 (0.65, 1.27)
79 0.98 (0.72, 1.34) 0.84 (0.59, 1.20)
0.81 0.54
76 1.0 1.0
125 86 62 1.39 (1.04, 1.85) 1.17 (0.86, 1.59) 1.06 (0.75, 1.48) 1.32 (0.97, 1.79) 1.06 (0.76, 1.48) 1.04 (0.72, 1.49)
0.99 0.78
47 1.0 1.0
38 0.67 (0.44, 1.03) 0.59 (0.37, 0.92)
62 0.98 (0.67, 1.43) 0.75 (0.49, 1.17)
46 0.85 (0.57, 1.28) 0.67 (0.42, 1.07)
0.83 0.22
43 1.0 1.0
74 47 36 1.38 (0.95, 2.01) 1.12 (0.74, 1.70) 1.15 (0.74, 1.79) 1.39 (0.93, 2.10) 1.02 (0.66, 1.60) 1.15 (0.71, 1.85)
0.76 0.94
24 1.0 1.0
34 1.39 (0.82, 2.34) 1.32 (0.77, 2.27)
32 1.30 (0.76, 2.21) 1.22 (0.69, 2.16)
30 1.27 (0.75, 2.18) 1.23 (0.68, 2.22)
0.50 0.66
30 1.0 1.0
39 24 29 1.44 (0.89, 2.31) 1.07 (0.64, 1.80) 0.87 (0.49, 1.53) 1.28 (0.77, 2.14) 0.93 (0.53, 1.63) 0.79 (0.43, 1.46)
0.43 0.27
362 47,479
445 46,460
513 45,498
614 43,863
31 1.0 1.0
33 1.04 (0.77, 1.41) 0.95 (0.56, 1.61)
41 1.22 (0.91, 1.64) 0.95 (0.54, 1.65)
49 1.15 (0.84, 1.57) 1.08 (0.57, 2.04)
0.25 0.74
30 1.0 1.0
48 40 36 1.31 (0.83, 2.07) 1.15 (0.72, 1.85) 1.05 (0.65, 1.71) 1.08 (0.64, 1.84) 0.91 (0.51, 1.64) 0.80 (0.40, 1.60)
0.97 0.39
59 1.0 1.0
75 1.07 (0.76, 1.50) 1.01 (0.70, 1.46)
93 1.11 (0.80, 1.54) 1.00 (0.67, 1.48)
101 1.14 (0.82, 1.57) 0.98 (0.62, 1.55)
0.44 0.92
74 1.0 1.0
108 81 86 1.13 (0.84, 1.52) 0.90 (0.66, 1.24) 1.01 (0.74, 1.38) 1.06 (0.75, 1.50) 0.85 (0.57, 1.25) 1.02 (0.65, 1.60)
0.73 0.84
32 1.0 1.0
37 0.88 (0.55, 1.42) 0.78 (0.47, 1.31)
61 1.13 (0.73, 1.74) 0.88 (0.52, 1.49)
63 1.07 (0.70, 1.65) 0.80 (0.43, 1.46)
0.51 0.61
38 1.0 1.0
61 50 51 1.16 (0.78, 1.75) 1.03 (0.68, 1.57) 1.16 (0.77, 1.77) 1.17 (0.73, 1.87) 1.04 (0.62, 1.74) 1.17 (0.64, 2.14)
0.61 0.76
27 1.0 1.0
32 1.13 (0.68, 1.88) 1.18 (0.67, 2.05)
26 0.87 (0.51, 1.50) 0.92 (0.49, 1.74)
35 1.16 (0.70, 1.92) 1.28 (0.63, 2.63)
0.73 0.60
28 1.0 1.0
44 29 21 1.11 (0.69, 1.79) 0.72 (0.42, 1.22) 0.86 (0.52, 1.43) 0.94 (0.53, 1.65) 0.60 (0.31, 1.16) 0.76 (0.36, 1.60)
0.32 0.34
307 57,548
388 56,478
442 56,721
525 56,305
1 Adjusted for age, energy, BMI, physical activity, smoking status, education, marital status, use of vitamin supplements and aspirin, and histories of diabetes and hypertension, and intakes of alcohol, SFAs, PUFAs, folate, salt, and fruits. For women, additionally adjusted for menopausal status.
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TABLE 2 HRs with 95% CIs for cardiovascular disease mortality by quartiles of betaine and choline intakes in Japanese men and women (1992–2008)
choline, further adjustments for major sources of betaine and choline intakes were made (Supplemental Tables 2 and 3). The results were not altered substantially. The sensitivity analyses revealed that the different methods estimating betaine intake did not affect the results substantially (Supplemental Table 4). The association between betaine intake and mortality from coronary heart disease as well as ischemic stroke in men did not differ significantly according to low and high intakes of folate, vitamin B-6, vitamin B-12, and Met (Supplemental Table 4). Exclusion of deaths during the first 4 y also did not alter the results substantially (Supplemental Table 4).
Discussion
Acknowledgments We thank Shougen Matsushita and Takehiko Minakuchi for their help in data collection. CN organized the study and wrote the manuscript; KW, TT, KK, TK, and MT conducted the data analysis and interpreted the data; KN helped supervise the field activities and interpret the data. All authors read and approved the final manuscript.
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Our study, as well as previous studies, observed no association between choline intake and cardiovascular disease (11, 12). The major fate of dietary choline is conversion to phosphatidylcholine (1). Phosphatidylcholine is essential for the secretion of VLDL from the liver (40). The TG-raising effect of phosphatidylcholine is suggested, which may contribute to negation of the inverse association with the risk of cardiovascular disease (41). Although intestinal microbial metabolism of dietary phosphatidylcholine has been implicated in the pathogenesis of atherosclerosis (10, 42), intake of choline from phosphatidylcholine was unrelated to mortality from coronary heart disease as well as stroke in our study. Because the bioavailability of different choline sources might differ, further studies on the association of intake of individual choline compounds with the risk of cardiovascular disease are warranted. Although our results were generally null, previous studies suggested that betaine could potentially influence cardiovascular disease risk. Several studies have found betaine to be a determinant for homocysteine concentration (2, 3, 6). Metaanalyses have shown that high homocysteine concentration is associated with the risk of coronary heart disease (7) and ischemic stroke (8). Although it is still unclear whether these relations are causal (43), it has been postulated that homocysteine may increase abnormal platelet activity and blood coagulation, which lead to thrombotic disorders (44). A high betaine intake may increase betaine availability and enhance remethylation of homocysteine (back to Met). Another study conducted among US men and women observed no association of betaine intake with the risk of coronary heart disease (11). The major source of dietary betaine is cereal foods in Western populations but is seafood in our population (45). Some studies have reported that high intake of seafood was associated with a decreased risk of coronary heart disease or ischemic stroke (46). Long-chain (n–3) PUFAs, which are mainly derived from fish and shellfish, are thought to be cardioprotective, antiarrhythmic, and antithrombotic (47). Although adjustment for seafood did not alter the present results, we cannot deny the possibility that long-chain (n–3) PUFAs or other components in fish or shellfish, rather than betaine, may be relevant to the risk of cardiovascular disease. On the other hand, the present study would suggest the possibility that betaine in seafood may be driving the association of seafood intake with cardiovascular disease. The strengths of the present study included its prospective design, good participation rate, representation of the general population, long follow-up, and information on potential confounders. Several limitations should be considered. It is known that seafood, especially marine invertebrates (10), is rich
in betaine. However, betaine data for invertebrates are scarce, probably because of the low consumption of shellfish in Western populations. Although adjustment for seafood intake did not alter the results and the sensitivity analyses assigning the different concentration values to shellfish did not alter the results substantially, measurement error could lead to biased estimates. Expansion of the reference database to include more seafood items is necessary. The sample size was limited, which precluded analyses on subtypes of stroke with small numbers of deaths. The use of mortality instead of incidence data prevented us from distinguishing the effect of diet on incidence, survival, or both. Our questionnaire was designed to measure an individualÕs relative intakes of nutrients rather than absolute values. Although we presented the mean values for nutrients in the table, some of them may be overestimated by our questionnaire. The mean values estimated from the FFQ were generally higher than those estimated from 12 daily diet records; e.g., the mean estimate of folate in the former was 19.7% higher in men and 14.8% higher in women than in the latter. Dietary habits measured only at baseline and the single assessment of dietary intake may not be reflective of long-term dietary exposure. Therefore, misclassification of betaine and choline intakes may have occurred because of measurement error and change of diet over time. The influence of underlying diseases or preclinical signs on dietary intake was possible, especially in the use of mortality data. In addition, we could not assess the very recent diet, which may have great impact on cardiovascular disease mortality with a very short induction time. Although numerous lifestyle and dietary factors were adjusted in the analyses, residual confounders or unmeasured variables could account for the observed associations. In conclusion, we found no clear evidence of significant associations between choline and betaine intakes and cardiovascular disease mortality in Japanese men and women. Our findings from subgroup analysis concerning betaine intake and mortality from coronary heart disease in men need confirmation in future studies because we cannot rule out that these findings could be caused by bias or chance.
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