Eur J Nutr DOI 10.1007/s00394-014-0729-5
ORIGINAL CONTRIBUTION
Low-dose B vitamins supplementation ameliorates cardiovascular risk: a double-blind randomized controlled trial in healthy Chinese elderly Linlin Wang • Hongtian Li • Yuan Zhou Lei Jin • Jianmeng Liu
•
Received: 24 November 2013 / Accepted: 2 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose We investigated whether daily supplementation with low-dose B vitamins in the healthy elderly population improves the Framingham risk score (FRS), a predictor of cardiovascular disease risk. Methods Between 2007 and 2012, a double-blind randomized controlled trial was conducted in a rural area of North China. In all, 390 healthy participants aged 60–74 were randomly allocated to receive daily vitamin C (50 mg; control group) or vitamin C plus B vitamins (400 lg folic acid, 2 mg B6, and 10 lg B12; treatment group) for 12 months. FRSs were calculated for all 390 subjects. Results Folate and vitamin B12 plasma concentrations in the treatment group increased by 253 and 80 %, respectively, after 6 months, stopped increasing with continued supplementation after 12 months and returned to baseline levels 6 months after supplementation cessation. Compared with the control group, there was no significant effect of B vitamin supplementation on FRSs after 6 months (mean difference -0.38; 95 % CI -1.06, 0.31; p = 0.279), whereas a significant effect of supplementation was evident after 12 months (reduced magnitude 7.6 %; -0.77; 95 % CI -1.47, -0.06; p = 0.033). However, this reduction
L. Wang H. Li L. Jin J. Liu (&) Institute of Reproductive and Child Health, Ministry of Health Key Laboratory of Reproductive Health, Department of Epidemiology and Biostatistics, School of Public Health, Peking University Health Science Centre, Beijing, People’s Republic of China e-mail: [email protected] Y. Zhou Xiacheng District Institute of Health Inspection, Hangzhou, People’s Republic of China
disappeared 6 months after supplementation stopped (-0.07; 95 % CI -0.80, 0.66; p = 0.855). The reduction in FRS 12 months after supplementation was more pronounced in individuals with a folate deficiency (10.4 %; -1.30; 95 % CI -2.54, -0.07; p = 0.039) than in those without (4.1 %; -0.38; 95 % CI -1.12, 0.36; p = 0.313). B vitamins increased high-density lipoprotein cholesterol by 3.4 % after 6 months (0.04; 95 % CI -0.02, 0.10; p = 0.155) and by 9.2 % after 12 months (0.11; 95 % CI 0.04, 0.18; p = 0.003). Compared with the control group, this change in magnitude decreased to 3.3 % (0.04; 95 % CI -0.02, 0.10; p = 0.194) 6 months after supplementation cessation. Conclusions Daily supplementation with a low-dose of B vitamins for 12 months reduced FRS, particularly in healthy elderly subjects with a folate deficiency. These reduced effects declined after supplementation cessation, indicating a need for persistent supplementation to maintain the associated benefits. Keywords B vitamins Cardiovascular disease risk Framingham risk score Prevention Abbreviations FRS Framingham risk score RDA Recommended dietary allowance
Introduction Plasma homocysteine is a well-recognized indicator of cardiovascular disease [1]. High homocysteine levels are associated with various factors including unhealthy
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lifestyle; deficiency in B vitamins such as folate, vitamin B6, and vitamin B12; the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T variant; and impaired renal function [2–4]. The effect of vitamin B supplements on reducing homocysteine concentrations has been well documented [5–7]. However, its effect on risk of cardiovascular disease remains controversial, and its usefulness in the elderly population is unknown. Several clinical trials have assessed the potential effect of vitamin B complex supplementation for the treatment of chronic cardiovascular disease in patients, yet most of these trials showed no apparent benefits [8–10]. However, observational studies have suggested that the consumption of foods fortified with folic acid or the dietary intake of vitamin B6 and B12 is associated with a decreased risk of cardiovascular disease [11, 12] in people with no previous history of cardiovascular disease, indicating the potential benefits of B vitamins in the primary prevention of cardiovascular events. Although meta-analyses of randomized controlled trials have shown potential mild benefits of folic acid supplementation in primary stroke prevention in people with cardiovascular disease, end-stage renal disease, and esophageal dysplasia [13, 14], the relationship between B vitamins and reduced risk of vascular disease in the general healthy population remains unknown. Furthermore, the qualified health claim on the daily intake of B vitamins from the U.S. Food and Drug Administration is controversial [15, 16]. Primary prevention trials among a general healthy population without a history of cardiovascular disease or other chronic diseases are needed to provide evidence for updating the government’s guideline for the general population. To conduct such a trial, it is more feasible to use appropriate surrogate endpoints than to use cardiovascular events in healthy elderly people, who are more likely to be deficient in B vitamins [17] and are thus at a higher risk of developing cardiovascular events [18]. The Framingham risk score (FRS), which is calculated on the basis of various classical risk factors for cardiovascular diseases, has been recommended for assessing the risk of cardiovascular events in asymptomatic patients or for screening high-risk people, according to National Cholesterol Education Program Adult Treatment Panel III guidelines in the United States [19]. Daily supplementation with high dosages of folic acid (2.5 mg), vitamin B6 (25–50 mg), and B12 (1 mg) in patients with cardiovascular or chronic kidney diseases has serious side effects, including impaired renal functions and increased risks of vascular events [9, 20]. Although considered safer than high dosages, low-dosage supplementation with B vitamins may still cause adverse effects when taken over the long term [21]. The goal of this study was to examine whether administration of B vitamins at levels close to the recommended dietary allowance (RDA) has
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beneficial effects on the risk of cardiovascular diseases in healthy elderly people. To this end, a randomized controlled trial was conducted to determine whether diets supplemented with B-complex vitamins at an approximate RDA dosage caused a decrease in FRSs in the healthy elderly population from a rural area of China where folic acid-fortified foods are unavailable. We hypothesized that dietary supplementation for 12 months with a low dose of B vitamins would reduce the FRS.
Subjects and methods Study design and participants This study was conducted from July 2007 through September 2012 in a rural county of Hebei province in North China, where gross domestic product per capita was about $3,500 in 2008. Eligible healthy elderly volunteers were enrolled from county healthcare hospitals; each volunteer was personally informed about the study’s purpose and methods before he or she was enrolled. Individuals were randomized to receive a daily supplement of vitamin C (control group) or vitamin C plus complex B vitamins (treatment group) for 12 months. We used vitamin C as a control because we wanted to provide the most health benefits to the participants in view of ethical considerations, and because vitamin C deficiency is common among the elderly in North China, who usually have little vegetables and fruits in their diets. Participants were followed-up monthly during the 12-month supplementation period, and 6 months after stopping supplementation. This study was approved by the Institutional Review Boards of Peking University Health Science Center (China). All participants provided written informed consent to participate in the study. The participants were aged 60–74 years and met the following inclusion criteria: (1) resided in the study area for [12 months; (2) were free of chronic diseases (i.e., cardiovascular disease, chronic obstructive pulmonary disease, diabetes, cancer, severe renal, and liver disease) and of severe mental disorders; (3) had not consumed any of the investigated vitamins (i.e., folic acid, B6, and B12) as supplements in the prior 6 months; (4) did not take any medication known to interfere with folate metabolism (i.e., methotrexate, tamoxifen, L-3,4-dihydroxyphenylalanine, niacin, phenytoin, bile acid sequestrants, dilantin, phenytoin, primidone, metformin, and sulfasalazine); and (5) gave written informed consent to participate in the study. Exclusion criteria included those who had chronic diseases and severe mental disorders, those who consumed any of the investigated vitamins in the previous 6 months, and those who took any medication known to interfere with
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folate metabolism. This trial was registered at clinicaltrials.gov as NCT00755664. Procedures Randomization was performed by a study statistician, who used a stratified randomization procedure. The stratification factors were age (60–64, 65–69, and 70–74 years) and gender (male/female), and the randomization list was generated in two steps using Microsoft Excel. First, simple randomization was implemented within each age and gender stratum in a 1:1 ratio using the RAND function. Second, 10 three-digit lot numbers were randomly assigned to the two supplement groups, with each set of five lot numbers representing one supplement. Aside from a pharmaceutical engineer, who ensured allocation of lot numbers to the correct supplement formulations, all investigators and participants were blinded to the identity of the supplements. Supplements were prepared in capsule form and were provided to participants at no cost. The capsules were not discernible in appearance, smell, taste, size, or packaging. Eligible participants were randomly allocated to receive a daily dose of 50 mg vitamin C or a combination of 400 lg folic acid, 2 mg vitamin B6, 10 lg vitamin B12, and 50 mg vitamin C. Before treatment, information on the participants’ sociodemographic characteristics and lifestyle was collected through face-to-face interviews. After randomization, participants were given a bottle of supplements containing 31 capsules and instructed to take one capsule per day for 12 months. Follow-up interviews were conducted every 4 weeks until supplementation was abated. During each interview, the number of capsules consumed was recorded to monitor compliance; the next bottle of supplements was given to the participants, and information on adverse events during the past 4 weeks was recorded. Collection of baseline information and subsequent followup visits were completed by trained project staff from the county healthcare hospital. Researchers from Peking University were responsible for project staff training, household visits (*5–10 participants were selected every month), and quality assurance. Venous fasting blood samples were collected in K3EDTA-containing vacutainer tubes after 6 and 12 months of supplementation, and 6 months after supplementation cessation at a local county healthcare hospital. All blood samples were immediately centrifuged at 3,000g for 15 min at 4 °C, and plasma and red blood cells were separated and frozen at -20 °C within 1 h of collection. The frozen samples were transported on dry ice to the laboratory at the Institute of Reproductive and Child Health (Peking University, China) and stored at -70 °C until the assays were performed.
Measurements FRSs were calculated for all study participants. Plasma folate, total homocysteine, vitamin B12, and lipid concentrations, as well as blood pressure, were determined after 6 and 12 months of supplementation, and 6 months after supplementation cessation. Plasma folate concentrations were assessed using a microbial assay (Lactobacillus casei) [22], and plasma concentrations of total homocysteine and vitamin B12 were measured using an immunofluorescence method (AxSYM system, Abbott Laboratories, Missisauga, Canada). Plasma total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were measured enzymatically (Sekisui Medical Co., Ltd, Tokyo, Japan). Additionally, baseline hemoglobin concentrations were determined using a colorimetric method (Horiba ABX Micros 60, ABX Diagnostic, Montpellier, France). In the laboratory, the intra- and inter-assay coefficients of variation for the assays were \9 % for folate, \8 % for homocysteine, \10 % for vitamin B12, and \5 % for plasma lipid and hemoglobin. Blood pressure was measured using a mercury sphygmomanometer according to the recommendations of the British Hypertension Society [23]. The 10-year risk assessment for determining 10-year risk was determined according to the FRS. The FRS risk factors, which were derived from an update of the Framingham database and methodology, were calculated based on age, total plasma cholesterol, HDL cholesterol, systolic blood pressure, and current smoking status (smoker vs. non-smoker) by a specific prediction algorithm [19, 24]. First, the number of points for each risk factor was calculated. Then, the points for each risk factor were added to obtain the total risk score. The 10-year risk for myocardial infarction, angina, and coronary death was estimated from the total risk score. Sample size To estimate the sample size of the trial, a pilot study was conducted in the same county of the Hebei province in North China in 2007. The mean plasma homocysteine concentration from the pilot study was 18 lmol/L (SD 10.7). Based on data from previous studies, we hypothesized that a 30 % reduction in plasma homocysteine was associated with B vitamin supplementation in each of the six age- and gender-specific strata [25]. With a two-sided significance level of 0.05 and a b-error specification of 0.20, 50 participants were needed to detect a 30 % reduction in the treatment group. Assuming a worst-case scenario of a 20 % dropout rate, 60 participants were needed for each stratum; thus, a total of 360 participants were needed. The pilot study showed that the mean FRS was 9.3
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Eur J Nutr Fig. 1 Flow of study participants
405 eligible participants 9 refused to offer blood 6 refused to receive vitamin 390 Underwent randomization Months Start 0
195 Assigned to treatment group
Supplementation
1 died 5 moved 9 Dropped out 6
180 blood sample collected and assayed 1 died 2 moved 4 Dropped out
Stopping Supplementation
Stop 12
18
173 blood sample collected and assayed 2 died 2 moved 3 Dropped out 166 blood sample collected and assayed
(SD 5.3). With a two-sided significance level of 0.05 and a sample size of 360, we have 80 % power to find a difference of 15 % of FRS between the two groups. Statistical analysis Compliance was assessed by the number of supplements consumed divided by the number of days from enrollment to the end of the intervention. We examined the randomization effectiveness by comparing the key baseline characteristics between the two groups. Quantitative variables were ln-transformed to achieve an approximate normal distribution. The chi-square test or independent samples t test was used to examine statistical differences between the two groups. The paired-samples t test was used to analyze changes in folate and B12 concentrations from baseline to 6, 12, and 18 months. Hyperhomocysteinemia was defined as homocysteine concentrations C16.0 lmol/L [26], and folate deficiency was defined as plasma folate concentrations B6.8 nmol/L [27]. The effect of intervention on the FRS was assessed using a general linear model and Fisher’s least significant difference test. Subgroup analyses were performed using a general linear model with adjustment for baseline FRS, baseline homocysteine, baseline folate, age, gender, education, current smoking,
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195 Assigned to control group 3 died 6 Dropped out 186 blood sample collected and assayed 1 died 3 moved 8 Dropped out 174 blood sample collected and assayed 2 died 3 moved 6 Dropped out 163 blood sample collected and assayed
alcohol consumption, BMI, and MTHFR C677T. The correlation between B vitamin supplementation and baseline folate or homocysteine concentrations was tested by adding a product term to the model. p values less than 0.05 were considered statistically significant. Statistical analyses were performed using the Statistical Package for the Social Sciences version 18.0 (SPSS Inc., Chicago, IL, USA).
Results Among the 405 potential eligible participants, 390 underwent randomization, 366 supplemented their diet for 6 months, 347 supplemented their diet for 12 months, and 329 completed the entire study, including the 6-month follow-up period after supplementation cessation (Fig. 1). Demographic characteristics were similar between the subjects who dropped out and those who participated in the entire study. No adverse events related to supplementation were reported during the study period. Baseline characteristics between the two groups were balanced (Table 1). The mean age, BMI, plasma folate, and plasma homocysteine concentrations were 66.8 years (SD 4.4), 23.5 kg/m2 (SD 3.7), 9.6 nmol/L (95 % CI 9.1, 10.1), and 19.8 lmol/L (95 % CI 18.8, 20.8), respectively.
Eur J Nutr Table 1 Baseline characteristics of the study population: elderly Chinese individuals aged 60–74 years (n = 390) Characteristics
Treatment group (n = 195)
Control group (n = 195)
p value*
Age, years, mean (SD)
66.7 (4.5)
66.8 (4.2)
0.72
Male (%)
49.2
50.8
0.76
Han ethnicity (%)
100.0
99.0
0.16
Farmer (%)
95.9
99.0
0.06
Illiteracy (%)
40.0
38.5
0.76
Current smoking (%)
27.2
24.6
0.56
Alcohol consumption (%)
15.4
15.9
0.89
Body mass index (kg/m2) mean (SD)
23.6 (3.9)
23.4 (3.5)
0.66
Hb, g/L, mean (SD)
149.5 (18.6)
149.9 (14.8)
0.81
Systolic blood pressure, mmHg, mean (SD)
133.2 (17.9)
131.2 (16.9)
0.25
Diastolic blood pressure, mmHg, mean (SD) Plasma folate, nmol/L, geometric mean (95 % CI)
79.3 (8.5) 9.9 (9.2, 10.6)
77.8 (8.3) 9.3 (8.7, 10.1)
0.07 0.30
Plasma folate deficiency (%)a
25.1
24.6
0.91
Red blood cell folate, nmol/L, geometric mean (95 % CI)
467.4 (436.4, 500.5)
466.7 (437.9, 497.3)
0.97
Plasma homocysteine concentrations, lmol/L, mean (95 % CI)
20.0 (18.6, 21.6)
19.5 (18.2, 20.9)
0.60
Hyperhomocysteinemia (%)b
59.5
62.1
0.60
Vitamin B12, pg/mL, geometric mean (95 % CI)c
189.4 (148.2, 242.1)
232.4 (202.2, 267.1)
0.16
Vitamin B12 deficiency (%)c
46.0
38.0
0.42
Total cholesterol, mmol/L, mean (SD)
4.22 (1.02)
4.23 (1.17)
0.89
High-density lipoprotein cholesterol, mmol/L, mean (SD)
1.17 (0.33)
1.19 (0.35)
0.54
Low-density lipoprotein cholesterol, mmol/L, mean (SD)
2.29 (0.74)
2.26 (0.76)
0.73
Triglyceride, mmol/L, mean (SD)
0.97 (0.42)
1.05 (0.41)
0.09
Framingham risk score, mean (95 % CI)
9.26 (8.30, 10.22)
9.01 (8.07, 9.95)
MTHFR C677T variant (%) CC
0.71 0.55
12.4
11.2
CT
49.7
45.3
TT
37.9
43.6
* p value from t test for quantitative variables and v2 test for qualitative variables a
Plasma folate deficiency was defined as plasma folate concentration B6.8 nmol/L
b
Hyperhomocysteinemia was defined as plasma homocysteine concentration C16.0 lmol/L
c
Plasma vitamin B12 deficiency was defined as plasma vitamin B12 concentration\203 pg/mL. Fifty samples from each group were assayed for plasma vitamin B12 concentrations
MTHFR 677TT genotype carriers represented 40.7 % of the study population. Almost all participants were of Han ethnicity and were farmers. The average compliance after 6 and 12 months of supplementation was 97 % and 90 %, respectively (both of which did not differ between the two groups). Plasma folate and B12 concentrations after 6, 12, and 18 months of supplementation are shown in Fig. 2. Compared with the control, the folate concentration in the treatment group increased by 253 % after 6 months (treatment group 38.6 nmol/L vs. control group 10.9 nmol/L; p \ 0.001) and by 172 % after 12 months (treatment group 37.2 nmol/L vs. control group 13.7 nmol/L; p \ 0.001). Vitamin B12 plasma concentrations increased by 80 % after 6 months (371 vs. 206 pg/mL; p \ 0.001) and 60 % after 12 months (343 vs. 214 pg/mL; p \ 0.001). Continued supplementation for 12 months
resulted in no further increase in folate or vitamin B12 concentrations compared to concentrations achieved at 6 months. Significant increases in folate concentrations (30 %, 17.2 vs. 13.2 nmol/L; p \ 0.001) and in vitamin B12 concentrations (31 %, 291 vs. 222 pg/mL; p = 0.043) were observed at 18 months, 6 months after supplementation cessation. Mean baseline homocysteine concentration was 20.0 (95 % CI 18.6, 21.6) lmol/L in the treatment and 19.5 (95 % CI 18.2, 20.9) lmol/L in the control (t = 0.519, p = 0.60). Compared with the control, supplementation with B vitamins reduced plasma homocysteine by 35.6 % (12.9 vs. 20.1 lmol/L; p \ 0.001) at 6 months after supplementation and by 43.4 % (mean difference -10.0 lmol/ L; 95 % CI -12.0, -8.1 lmol/L; p \ 0.001) at 12 months after supplementation.
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Plasma folate b
50
Geometric mean
*** ‡
40
*** ‡
30 *** ‡
20 10 0
0
6
12
18
Time after randomization, months Fig. 2 Folate (a) and vitamin B12 (b) concentrations after randomization (supplementation stopped after 12 months). Error bars indicate 95 % confidence intervals. Values were ln-transformed before
Geometric mean
a
Vitamin B12 treatment
500
*** ‡
*** ‡
400
control *** †
300 200 100 0
0
6
12
18
Time after randomization, months analysis. Compared with baseline, paired-samples t test in treatment group, ***p \ 0.001. Compared with the control group, independent samples t test, àp \ 0.001, p \ 0.05
Table 2 Framingham risk score on coronary heart disease after randomization (supplementation stopped after 12 months) n Control
Differencea
Mean (95 % CI)* Treatment
%b
Control group
Treatment group
Mean (95 % CI)
p value
Framingham risk score (unadjusted) 6 months 12 months
183 169
179 170
10.17 (9.19, 11.14) 10.01 (8.97, 11.04)
10.10 (9.11, 11.08) 9.41 (8.38, 10.44)
-0.07 (-1.46, 1.31) -0.59 (-2.06, 0.87)
0.916 0.425
-0.7 -5.9
18 months
156
158
9.72 (8.68, 10.77)
9.49 (8.45, 10.54)
-0.23 (-1.71, 1.25)
0.759
-2.4
Framingham risk score (adjusted)c 6 months
183
179
10.31 (9.83, 10.80)
9.94 (9.45, 10.42)
-0.38 (-1.06, 0.31)
0.279
-3.7
12 months
169
170
10.10 (9.60, 10.59)
9.33 (8.83, 9.83)
-0.77 (-1.47, -0.06)
0.033
-7.6
18 months
156
158
9.62 (9.11, 10.14)
9.55 (9.04, 10.07)
-0.07 (-0.80, 0.66)
0.855
-0.7
* Compared with baseline, the FRS at 6, 12, and 18 months increased in both the control and treatment groups (p \ 0.05) a
Difference between the treatment and placebo groups was determined by a general linear model and the Fisher’s least significant difference test, 95 % CI is shown in parentheses
b
Percent value = 100 9 difference of mean between treatment and control/mean of control
c
Values are estimated marginal means adjusted for baseline FRS
Mean baseline FRS was 9.26 (95 % CI 8.30, 10.22) in the treatment group and 9.01 (95 % CI 8.07, 9.95) in the control group (p = 0.71) (Table 1). Stratified analysis showed no difference in baseline FRS between the treatment and control groups, as determined by evaluating folate and vitamin B12 concentrations. Compared with baseline concentrations, the FRS after 6, 12, and 18 months increased in both the control and treatment groups (p \ 0.05). Compared with the control group, there was no significant effect of B vitamin supplements on the FRS after 6 months (mean difference -0.38; 95 % CI -1.06, 0.31; p = 0.279), whereas a significant effect of supplementation was evident after 12 months (reduced magnitude 7.6 %; -0.77; 95 % CI -1.47, -0.06; p = 0.033), but this reduction disappeared 6 months after supplementation cessation (-0.07; 95 % CI -0.80, 0.66; p = 0.855) (Table 2). We further observed that the reduction effects existed in all strata classified by baseline folate or homocysteine
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concentration, and the magnitude of this reduction was more pronounced in people with baseline folate deficiency (2.9 % at 6 months; 10.4 % at 12 months) or hyperhomocysteinemia (2.8 % at 6 months; 7.5 % at 12 months) (Table 3). Similar subgroup results were also observed 6 months after the study participants stopped supplementing their diet. The main components of the FRS were examined after 6 and 12 months of supplementation, and 6 months after stopping supplementation (Table 4). Mean baseline HDL cholesterol was 1.17 (95 % CI 1.12, 1.22) in the treatment group and 1.19 (95 % CI 1.14, 1.24) in the control group (p = 0.542). Compared with the control, supplementation for 6 months showed a trend toward increasing HDL cholesterol by 3.4 % (0.04; 95 % CI -0.02, 0.10; p = 0.155), and supplementation for 12 months significantly increased HDL cholesterol by 9.2 % (0.11; 95 % CI 0.04, 0.18; p = 0.003); the magnitude of change decreased to 3.3 % (0.04; 95 % CI -0.02, 0.10; p = 0.194) 6 months
Eur J Nutr Table 3 Stratified analysis for FRS after randomization (supplementation stopped after 12 months) n
Differencea
Mean (95 % CI)*
Control
Treatment
%b
Control
Treatment
Mean (95 % CI)
p value
Baseline folate levelc B6.8 nmol/L 6 months
42
43
12.54 (11.38, 13.72)
12.19 (11.03, 13.34)
-0.36 (-2.08, 1.37)
0.682
-2.9
12 months
40
40
12.50 (11.66, 13.34)
11.20 (10.36, 12.04)
-1.30 (-2.54, -0.07)
0.039
-10.4
18 months
34
36
12.54 (11.38, 13.70)
11.60 (10.48, 12.72)
-0.94 (-2.62, 0.74)
0.265
-7.5
6 months 12 months
141 129
135 129
9.29 (8.79, 9.79) 9.16 (8.64, 9.68)
9.26 (8.75, 9.77) 8.78 (8.26,9.30)
-0.03 (-0.75, 0.69) -0.38 (-1.12, 0.36)
0.935 0.313
-0.3 -4.1
18 months
122
121
8.67 (8.11, 9.23)
8.95 (8.40, 9.50)
0.28 (-0.51, 1.06)
0.491
3.2
[6.8 nmol/L
Baseline homocysteine levelc \16.0 lmol/L 6 months
72
71
8.41 (7.75, 9.07)
8.33 (7.69, 8.98)
-0.07 (-1.01, 0.86)
0.875
-0.8
12 months
64
68
8.43 (7.73, 9.13)
7.82 (7.16, 8.47)
-0.61 (-1.59, 0.36)
0.214
-7.2
18 months
60
63
8.05 (7.30, 8.80)
7.91 (7.20, 8.61)
-0.14 (-1.19, 0.90)
0.786
-1.7
6 months
111
107
11.25 (10.61, 11.89)
10.93 (10.28, 11.59)
-0.32 (-1.25, 0.61)
0.497
-2.8
12 months
105
101
11.05 (10.49, 11.62)
10.23 (9.65, 10.81)
-0.83 (-1.64, -0.01)
0.048
-7.5
18 months
96
94
10.56 (9.88, 11.24)
10.57 (9.88, 11.27)
0.02 (-0.97, 1.00)
0.976
0.2
C16.0 lmol/L
* Values were estimated by marginal means adjusted for baseline FRS, baseline homocysteine level, baseline folate level, age, gender, education, current smoking, alcohol consumption, body mass index, and MTHFR C677T; 95 % CI is shown in parentheses a
Difference between the treatment and placebo groups was determined by a general linear model with adjustment for the variables listed above and by the Fisher’s least significant difference test
b
Percent value = 100 9 difference of mean between treatment and control/adjusted mean of control
c
No significant interaction was found between treatment and baseline folate levels or baseline homocysteine levels using a general linear model with adjustment for the variables listed above
Table 4 Main components of FRS on coronary heart disease after randomization (supplementation stopped after 12 months) n Control
Differencea
Mean (95 % CI)* Treatment
Control
Treatment
Mean (95 % CI)
%b p value
High-density lipoprotein cholesterol (mmol/L) 6 months
183
179
1.19 (1.14, 1.23)
1.23 (1.19, 1.27)
0.04 (-0.02, 0.10)
0.155
3.4
12 months
169
170
1.17 (1.12, 1.22)
1.27 (1.22, 1.32)
0.11 (0.04, 0.18)
0.003
9.2
18 months
156
158
1.20 (1.16, 1.24)
1.24 (1.20, 1.28)
0.04 (-0.02, 0.10)
0.194
3.3
Total plasma cholesterol (mmol/L) 6 months
183
179
4.48 (4.36, 4.61)
4.42 (4.29, 4.55)
-0.06 (-0.24, 0.12)
0.499
-1.3
12 months 18 months
169 156
170 158
4.01 (3.88, 4.14) 4.32 (4.20, 4.43)
4.01 (3.89, 4.14) 4.38 (4.27, 4.49)
0.00 (-0.17, 0.18) 0.06 (-0.10, 0.22)
0.960 0.452
0.0 0.0
Systolic blood pressure (mmHg) 6 months
183
179
140.6 (138.0, 143.3)
142.5 (139.8, 145.2)
1.86 (-1.91, 5.64)
0.332
1.3
12 months
169
170
134.5 (131.9, 137.0)
133.6 (131.0, 136.2)
-0.89 (-4.50, 2.72)
0.628
-0.7
18 months
156
158
139.8 (137.2, 142.4)
138.7 (136.2, 141.3)
-1.04 (-4.66, 2.58)
0.573
-0.7
* Values are estimated marginal means adjusted for baseline level of each component a
Difference between the treatment and placebo groups was determined using a general linear model and by the Fisher’s least significant difference test; 95 % CI is shown in parentheses b Percent value = 100 9 difference of mean between treatment and control/mean of control
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after stopping supplementation. No significant change was found in total plasma cholesterol or systolic blood pressure.
Discussion In this randomized controlled trial, we found that daily supplementation with a low dosage of folic acid, vitamin B6, and vitamin B12 for 12 months effectively reduced the FRS, particularly in healthy elderly people with a folate deficiency. We also found that B vitamins increased HDL cholesterol after 12 months. The magnitude of reduction in the FRS and HDL cholesterol returned to baseline after 6 months of supplementation cessation. Despite the fact that the FRS has been used as a primary outcome measure in trials to investigate the effects of nutraceutical supplements [28] and fruit and vegetable intake [29], to date, no trial has examined the effects of low-dose complex B vitamins on the FRS. Here, we found that 12-month supplementation with B vitamins reduced the FRS by 7.6 % overall and by 11 % in folate-deficient elderly people compared to the control group, although the FRS at 12 months increased in both the control and treatment groups with age compared with baseline. The reduction in magnitude of the FRS declined after supplementation cessation, further indicating a need for persistent supplementation to maintain the associated benefits. Additionally, the participants in our clinical trial did not encounter any serious adverse events related to the supplementation, indicating the feasibility of potential universal supplementation with B vitamins near the RDA in healthy elderly individuals. Although no trial has investigated the effects of lowdose B-complex vitamins on the FRS in healthy adults, some trials have investigated the effects of high-dose B vitamins on other functional (e.g., exercise electrocardiography testing) [30] or morphological indicators (e.g., carotid arterial stiffness and carotid intima-media thickness) of cardiovascular diseases [5]. Indeed, previous trials like the one described in this report have shown the beneficial effects on functional indicators [30], but not for morphological markers [5]. This is most likely due to a long imperceptible process in the development of morphological changes [31], which may further highlight the importance of prophylactic supplementation of B vitamins in the primary prevention of cardiovascular diseases. The mechanism underlying the effect of B vitamins on improving functional indicators is possibly related to their role in promoting the re-methylation of homocysteine [32], scavenging superoxide radicals [33], and/or advancing blood lipid metabolism [34]. We found that plasma HDL cholesterol significantly increased by 9.2 % 12 months after supplementation with B vitamins; however, this
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elevation decreased after supplementation ceased. Similar patterns were observed between changes in FRS and plasma HDL cholesterol, indicating that advancing blood lipid metabolism might be one of the major mechanisms underlying the beneficial effect of B vitamins on cardiovascular diseases. Our findings are in accordance with previous studies, which demonstrated that folic acid supplementation in healthy postmenopausal Italian women increased HDL cholesterol levels by 6 and 12 % after 1 month and 8 weeks, respectively, of folic acid administration (7.5 mg/day) [34, 35]. Additionally, an observational study in young healthy non-smokers showed that plasma folate levels were associated with HDL cholesterol levels and endothelial function in the brachial artery, regardless of homocysteine status, indicating that low folate levels may be an independent risk factor for cardiovascular diseases [36]. The mechanism underlying the correlation between B vitamin supplementation and increased HDL cholesterol levels might be related to improvement of hepatic metabolism and antioxidation [34, 35]. Increased HDL cholesterol can protect endothelial function by stimulating reverse cholesterol transport, antioxidant, anti-apoptotic, and/or anti-inflammatory properties, all of which are beneficial for cardiovascular health [37]. In this randomized controlled trial, we also found that both folate and vitamin B12 concentrations in the treatment group increased and reached a high level after 6 months of supplementation, stopped increasing with continued supplementation for a total of 12 months, and returned to baseline after 6 months of supplementation cessation. A similar change in pattern was reported by Crider et al. [38], who demonstrated that the highest level of folate was achieved 3 months or 6 months after supplementation of folic acid at doses of 100, 400, and 4,000 lg/day and 4,000 lg/week in Northern Chinese women of childbearing age; a significant decrease was observed after 3 months of supplementation cessation in all dose groups, regardless of MTHFR genotype [38]. Therefore, it appears that there is a plateau for increased plasma folate and vitamin B12 level after 3 or 6 months of supplementation, and the intervention-enhanced folate status rapidly diminishes once supplementation ceases. Our trial had several strengths. First, the dosage of B vitamins was close to the RDA [39] and did not cause serious adverse effects. This is important, as an overdose of folic acid can promote cell proliferation, raise asymmetric dimethylarginine levels, and affect pro-atherogenic gene expression, which might induce unexpected toxicity [40, 41]. Second, the fortification of foods with folic acid was not available in our study area, and folate and vitamin B12 deficiency was prevalent in our study population, which provided us with the opportunity to examine the benefits of
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B vitamins at a low dosage. In several previous large-scale trials, folate fortification and high baseline folate levels in the study areas might have contributed to the negative results of the effects of B vitamins on the prevention of cardiovascular events [8–10]. Despite these strengths, it should be noted that the FRS has not been well validated in the Chinese population, which may affect the validity of our findings. However, the FRS is calculated based on several well-accepted classic risk factors for cardiovascular diseases, indicating that our findings are most likely valid. In conclusion, daily supplementation with low-dose B vitamins for 12 months reduced the FRS, especially in healthy elderly people with a folate deficiency. The benefits diminished after supplementation cessation, indicating a need for the persistent use of low-dose B vitamins to maintain the associated benefits. Future work is warranted to examine whether the results are applicable to people in other societies or in Chinese people less than 60 years of age.
9.
10.
11.
12.
13.
14.
15. Acknowledgments This study was supported by two grants from the National Natural Science Foundation of China (Grant Nos. 30572071; 30471486). We thank Dr. Zuguo Mei (U.S. Centers for Disease Control and Prevention) and Dr. Shufeng Zhou (University of South Florida) for their thoughtful review and editing of this manuscript. We thank all participants for their dedication and the local health workers for their assistance with subject recruitment and data collection.
16.
17. Conflict of interest
The authors declare no conflicts of interest.
References 18. 1. Ueland PM, Loscalzo J (2012) Homocysteine and cardiovascular risk: the perils of reductionism in a complex system. Clin Chem 58:1623–1625 2. McCully KS (2007) Homocysteine, vitamins, and vascular disease prevention. Am J Clin Nutr 86:1563S–1568S 3. Hustad S, Midttun O, Schneede J, Vollset SE, Grotmol T, Ueland PM (2007) The methylenetetrahydrofolate reductase 677C?T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism. Am J Hum Genet 80:846–855 4. Robinson K (2004) Renal disease, homocysteine, and cardiovascular complications. Circulation 109:294–295 5. Durga J, Bots ML, Schouten EG, Grobbee DE, Kok FJ, Verhoef P (2011) Effect of 3 y of folic acid supplementation on the progression of carotid intima-media thickness and carotid arterial stiffness in older adults. Am J Clin Nutr 93:941–949 6. Armitage JM, Bowman L, Clarke RJ, Wallendszus K, Bulbulia R, Rahimi K, Haynes R, Parish S, Sleight P, Peto R, Collins R (2010) Effects of homocysteine-lowering with folic acid plus vitamin B12 vs. placebo on mortality and major morbidity in myocardial infarction survivors: a randomized trial. JAMA 303:2486–2494 7. Clarke R, Halsey J, Bennett D, Lewington S (2011) Homocysteine and vascular disease: review of published results of the homocysteine-lowering trials. J Inherit Metab Dis 34:83–91 8. Bonaa KH, Njolstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, Wang H, Nordrehaug JE, Arnesen E, Rasmussen K
19.
20.
21.
22.
23.
24.
(2006) Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 354:1578–1588 Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, McQueen MJ, Probstfield J, Fodor G, Held C, Genest J Jr (2006) Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 354:1567–1577 The VITATOPS Trial Study Group (2010) B vitamins in patients with recent transient ischaemic attack or stroke in the VITAmins TO Prevent Stroke (VITATOPS) trial: a randomised, doubleblind, parallel, placebo-controlled trial. Lancet Neurol 9:855–865 Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ (1998) Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279:359–364 Voutilainen S, Rissanen TH, Virtanen J, Lakka TA, Salonen JT (2001) Low dietary folate intake is associated with an excess incidence of acute coronary events: the Kuopio Ischemic Heart Disease Risk Factor Study. Circulation 103:2674–2680 Lee M, Hong KS, Chang SC, Saver JL (2010) Efficacy of homocysteine-lowering therapy with folic acid in stroke prevention: a meta-analysis. Stroke 41:1205–1212 Wang X, Qin X, Demirtas H, Li J, Mao G, Huo Y, Sun N, Liu L, Xu X (2007) Efficacy of folic acid supplementation in stroke prevention: a meta-analysis. Lancet 369:1876–1882 US Food and Drug Administration (2000) Letter regarding dietary supplement health claim for folic acid, vitamin B6, and vitamin B12 and vascular disease (Docket No. 99P-3029). http:// www.fda.gov/Food/IngredientsPackagingLabeling/LabelingNutri tion/ucm072865.htm#tab US Food and Drug Administration (2001) Settlement reached for health claim relating B vitamins and vascular disease. http:// www.fda.gov/Food/IngredientsPackagingLabeling/LabelingNutri tion/ucm072855.htm Elshorbagy AK, Oulhaj A, Konstantinova S, Nurk E, Ueland PM, Tell GS, Nygard O, Vollset SE, Refsum H (2007) Plasma creatinine as a determinant of plasma total homocysteine concentrations in the Hordaland Homocysteine Study: use of statistical modeling to determine reference limits. Clin Biochem 40:1209–1218 Gale CR, Ashurst H, Phillips NJ, Moat SJ, Bonham JR, Martyn CN (2001) Renal function, plasma homocysteine and carotid atherosclerosis in elderly people. Atherosclerosis 154:141–146 National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (2002) Third report of the National Cholesterol Education Program (NCEP) expert panel on detection E, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 106:3143–3421 House AA, Eliasziw M, Cattran DC, Churchill DN, Oliver MJ, Fine A, Dresser GK, Spence JD (2010) Effect of B-vitamin therapy on progression of diabetic nephropathy: a randomized controlled trial. JAMA 303:1603–1609 Tighe P, Ward M, McNulty H, Finnegan O, Dunne A, Strain J, Molloy AM, Duffy M, Pentieva K, Scott JM (2011) A dosefinding trial of the effect of long-term folic acid intervention: implications for food fortification policy. Am J Clin Nutr 93:11–18 O’Broin S, Kelleher B (1992) Microbiological assay on microtitre plates of folate in serum and red cells. J Clin Pathol 45:344–347 Petrie JC, O’Brien ET, Littler WA, de Swiet M (1986) Recommendations on blood pressure measurement. Br Med J (Clin Res Ed) 293:611–615 Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB (1998) Prediction of coronary heart disease using risk factor categories. Circulation 97:1837–1847
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Eur J Nutr 25. Collaboration Homocysteine Lowering Trialists’ (2005) Dosedependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials. Am J Clin Nutr 82:806–812 26. Ueland PM, Refsum H, Stabler SP, Malinow MR, Andersson A, Allen RH (1993) Total homocysteine in plasma or serum: methods and clinical applications. Clin Chem 39:1764–1779 27. Federation of American Societies for Experimental Biology (1984) Assessment of the folate nutritional status of the U.S. population based on data collected in the second National Health and Nutrition Examination Survey, 1976–1980. Federation of American Societies for Experimental Biology, Bethesda, MD 28. Izzo R, de Simone G, Giudice R, Chinali M, Trimarco V, De Luca N, Trimarco B (2010) Effects of nutraceuticals on prevalence of metabolic syndrome and on calculated Framingham Risk Score in individuals with dyslipidemia. J Hypertens 28:1482–1487 29. Bhupathiraju SN, Tucker KL (2011) Greater variety in fruit and vegetable intake is associated with lower inflammation in Puerto Rican adults. Am J Clin Nutr 93:37–46 30. Vermeulen EG, Stehouwer CD, Twisk JW, van den Berg M, de Jong SC, Mackaay AJ, van Campen CM, Visser FC, Jakobs CA, Bulterjis EJ, Rauwerda JA (2000) Effect of homocysteine-lowering treatment with folic acid plus vitamin B6 on progression of subclinical atherosclerosis: a randomised, placebo-controlled trial. Lancet 355:517–522 31. Morrison KM, Atkinson SA, Yusuf S, Bourgeois J, McDonald S, McQueen MJ, Persadie R, Hunter B, Pogue J, Teo K (2009) The Family Atherosclerosis Monitoring In early life (FAMILY) study: rationale, design, and baseline data of a study examining the early determinants of atherosclerosis. Am Heart J 158:533–539 32. Wilcken DE, Dudman NP, Tyrrell PA, Robertson MR (1988) Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: possible implications for prevention of vascular disease. Metabolism 37:697–701
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33. Verhaar MC, Stroes E, Rabelink TJ (2002) Folates and cardiovascular disease. Arterioscler Thromb Vasc Biol 22:6–13 34. Villa P, Perri C, Suriano R, Cucinelli F, Panunzi S, Ranieri M, Mele C, Lanzone A (2005) L-folic acid supplementation in healthy postmenopausal women: effect on homocysteine and glycolipid metabolism. J Clin Endocrinol Metab 90:4622–4629 35. Paradisi G, Cucinelli F, Mele MC, Barini A, Lanzone A, Caruso A (2004) Endothelial function in post-menopausal women: effect of folic acid supplementation. Hum Reprod 19:1031–1035 36. Imamura A, Murakami R, Takahashi R, Cheng XW, Numaguchi Y, Murohara T, Okumura K (2010) Low folate levels may be an atherogenic factor regardless of homocysteine levels in young healthy nonsmokers. Metabolism 59:728–733 37. Tran-Dinh A, Diallo D, Delbosc S, Varela-Perez LM, Dang QB, Lapergue B, Burillo E, Michel JB, Levoye A, Martin-Ventura JL, Meilhac O (2013) HDL and endothelial protection. Br J Pharmacol 169:493–511 38. Crider KS, Zhu JH, Hao L, Yang QH, Yang TP, Gindler J, Maneval DR, Quinlivan EP, Li Z, Bailey LB, Berry RJ (2011) MTHFR 677C?T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation. Am J Clin Nutr 93:1365–1372 39. Office of Dietary Supplements National Institutes of Health (2013) Dietary supplement fact sheet. http://ods.od.nih.gov/fact sheets/list-all/ 40. Antoniades C, Antonopoulos AS, Tousoulis D, Marinou K, Stefanadis C (2009) Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials. Eur Heart J 30:6–15 41. Loscalzo J (2006) Homocysteine trials—clear outcomes for complex reasons. N Engl J Med 354:1629–1632
ORIGINAL CONTRIBUTION
Low-dose B vitamins supplementation ameliorates cardiovascular risk: a double-blind randomized controlled trial in healthy Chinese elderly Linlin Wang • Hongtian Li • Yuan Zhou Lei Jin • Jianmeng Liu
•
Received: 24 November 2013 / Accepted: 2 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose We investigated whether daily supplementation with low-dose B vitamins in the healthy elderly population improves the Framingham risk score (FRS), a predictor of cardiovascular disease risk. Methods Between 2007 and 2012, a double-blind randomized controlled trial was conducted in a rural area of North China. In all, 390 healthy participants aged 60–74 were randomly allocated to receive daily vitamin C (50 mg; control group) or vitamin C plus B vitamins (400 lg folic acid, 2 mg B6, and 10 lg B12; treatment group) for 12 months. FRSs were calculated for all 390 subjects. Results Folate and vitamin B12 plasma concentrations in the treatment group increased by 253 and 80 %, respectively, after 6 months, stopped increasing with continued supplementation after 12 months and returned to baseline levels 6 months after supplementation cessation. Compared with the control group, there was no significant effect of B vitamin supplementation on FRSs after 6 months (mean difference -0.38; 95 % CI -1.06, 0.31; p = 0.279), whereas a significant effect of supplementation was evident after 12 months (reduced magnitude 7.6 %; -0.77; 95 % CI -1.47, -0.06; p = 0.033). However, this reduction
L. Wang H. Li L. Jin J. Liu (&) Institute of Reproductive and Child Health, Ministry of Health Key Laboratory of Reproductive Health, Department of Epidemiology and Biostatistics, School of Public Health, Peking University Health Science Centre, Beijing, People’s Republic of China e-mail: [email protected] Y. Zhou Xiacheng District Institute of Health Inspection, Hangzhou, People’s Republic of China
disappeared 6 months after supplementation stopped (-0.07; 95 % CI -0.80, 0.66; p = 0.855). The reduction in FRS 12 months after supplementation was more pronounced in individuals with a folate deficiency (10.4 %; -1.30; 95 % CI -2.54, -0.07; p = 0.039) than in those without (4.1 %; -0.38; 95 % CI -1.12, 0.36; p = 0.313). B vitamins increased high-density lipoprotein cholesterol by 3.4 % after 6 months (0.04; 95 % CI -0.02, 0.10; p = 0.155) and by 9.2 % after 12 months (0.11; 95 % CI 0.04, 0.18; p = 0.003). Compared with the control group, this change in magnitude decreased to 3.3 % (0.04; 95 % CI -0.02, 0.10; p = 0.194) 6 months after supplementation cessation. Conclusions Daily supplementation with a low-dose of B vitamins for 12 months reduced FRS, particularly in healthy elderly subjects with a folate deficiency. These reduced effects declined after supplementation cessation, indicating a need for persistent supplementation to maintain the associated benefits. Keywords B vitamins Cardiovascular disease risk Framingham risk score Prevention Abbreviations FRS Framingham risk score RDA Recommended dietary allowance
Introduction Plasma homocysteine is a well-recognized indicator of cardiovascular disease [1]. High homocysteine levels are associated with various factors including unhealthy
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lifestyle; deficiency in B vitamins such as folate, vitamin B6, and vitamin B12; the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T variant; and impaired renal function [2–4]. The effect of vitamin B supplements on reducing homocysteine concentrations has been well documented [5–7]. However, its effect on risk of cardiovascular disease remains controversial, and its usefulness in the elderly population is unknown. Several clinical trials have assessed the potential effect of vitamin B complex supplementation for the treatment of chronic cardiovascular disease in patients, yet most of these trials showed no apparent benefits [8–10]. However, observational studies have suggested that the consumption of foods fortified with folic acid or the dietary intake of vitamin B6 and B12 is associated with a decreased risk of cardiovascular disease [11, 12] in people with no previous history of cardiovascular disease, indicating the potential benefits of B vitamins in the primary prevention of cardiovascular events. Although meta-analyses of randomized controlled trials have shown potential mild benefits of folic acid supplementation in primary stroke prevention in people with cardiovascular disease, end-stage renal disease, and esophageal dysplasia [13, 14], the relationship between B vitamins and reduced risk of vascular disease in the general healthy population remains unknown. Furthermore, the qualified health claim on the daily intake of B vitamins from the U.S. Food and Drug Administration is controversial [15, 16]. Primary prevention trials among a general healthy population without a history of cardiovascular disease or other chronic diseases are needed to provide evidence for updating the government’s guideline for the general population. To conduct such a trial, it is more feasible to use appropriate surrogate endpoints than to use cardiovascular events in healthy elderly people, who are more likely to be deficient in B vitamins [17] and are thus at a higher risk of developing cardiovascular events [18]. The Framingham risk score (FRS), which is calculated on the basis of various classical risk factors for cardiovascular diseases, has been recommended for assessing the risk of cardiovascular events in asymptomatic patients or for screening high-risk people, according to National Cholesterol Education Program Adult Treatment Panel III guidelines in the United States [19]. Daily supplementation with high dosages of folic acid (2.5 mg), vitamin B6 (25–50 mg), and B12 (1 mg) in patients with cardiovascular or chronic kidney diseases has serious side effects, including impaired renal functions and increased risks of vascular events [9, 20]. Although considered safer than high dosages, low-dosage supplementation with B vitamins may still cause adverse effects when taken over the long term [21]. The goal of this study was to examine whether administration of B vitamins at levels close to the recommended dietary allowance (RDA) has
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beneficial effects on the risk of cardiovascular diseases in healthy elderly people. To this end, a randomized controlled trial was conducted to determine whether diets supplemented with B-complex vitamins at an approximate RDA dosage caused a decrease in FRSs in the healthy elderly population from a rural area of China where folic acid-fortified foods are unavailable. We hypothesized that dietary supplementation for 12 months with a low dose of B vitamins would reduce the FRS.
Subjects and methods Study design and participants This study was conducted from July 2007 through September 2012 in a rural county of Hebei province in North China, where gross domestic product per capita was about $3,500 in 2008. Eligible healthy elderly volunteers were enrolled from county healthcare hospitals; each volunteer was personally informed about the study’s purpose and methods before he or she was enrolled. Individuals were randomized to receive a daily supplement of vitamin C (control group) or vitamin C plus complex B vitamins (treatment group) for 12 months. We used vitamin C as a control because we wanted to provide the most health benefits to the participants in view of ethical considerations, and because vitamin C deficiency is common among the elderly in North China, who usually have little vegetables and fruits in their diets. Participants were followed-up monthly during the 12-month supplementation period, and 6 months after stopping supplementation. This study was approved by the Institutional Review Boards of Peking University Health Science Center (China). All participants provided written informed consent to participate in the study. The participants were aged 60–74 years and met the following inclusion criteria: (1) resided in the study area for [12 months; (2) were free of chronic diseases (i.e., cardiovascular disease, chronic obstructive pulmonary disease, diabetes, cancer, severe renal, and liver disease) and of severe mental disorders; (3) had not consumed any of the investigated vitamins (i.e., folic acid, B6, and B12) as supplements in the prior 6 months; (4) did not take any medication known to interfere with folate metabolism (i.e., methotrexate, tamoxifen, L-3,4-dihydroxyphenylalanine, niacin, phenytoin, bile acid sequestrants, dilantin, phenytoin, primidone, metformin, and sulfasalazine); and (5) gave written informed consent to participate in the study. Exclusion criteria included those who had chronic diseases and severe mental disorders, those who consumed any of the investigated vitamins in the previous 6 months, and those who took any medication known to interfere with
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folate metabolism. This trial was registered at clinicaltrials.gov as NCT00755664. Procedures Randomization was performed by a study statistician, who used a stratified randomization procedure. The stratification factors were age (60–64, 65–69, and 70–74 years) and gender (male/female), and the randomization list was generated in two steps using Microsoft Excel. First, simple randomization was implemented within each age and gender stratum in a 1:1 ratio using the RAND function. Second, 10 three-digit lot numbers were randomly assigned to the two supplement groups, with each set of five lot numbers representing one supplement. Aside from a pharmaceutical engineer, who ensured allocation of lot numbers to the correct supplement formulations, all investigators and participants were blinded to the identity of the supplements. Supplements were prepared in capsule form and were provided to participants at no cost. The capsules were not discernible in appearance, smell, taste, size, or packaging. Eligible participants were randomly allocated to receive a daily dose of 50 mg vitamin C or a combination of 400 lg folic acid, 2 mg vitamin B6, 10 lg vitamin B12, and 50 mg vitamin C. Before treatment, information on the participants’ sociodemographic characteristics and lifestyle was collected through face-to-face interviews. After randomization, participants were given a bottle of supplements containing 31 capsules and instructed to take one capsule per day for 12 months. Follow-up interviews were conducted every 4 weeks until supplementation was abated. During each interview, the number of capsules consumed was recorded to monitor compliance; the next bottle of supplements was given to the participants, and information on adverse events during the past 4 weeks was recorded. Collection of baseline information and subsequent followup visits were completed by trained project staff from the county healthcare hospital. Researchers from Peking University were responsible for project staff training, household visits (*5–10 participants were selected every month), and quality assurance. Venous fasting blood samples were collected in K3EDTA-containing vacutainer tubes after 6 and 12 months of supplementation, and 6 months after supplementation cessation at a local county healthcare hospital. All blood samples were immediately centrifuged at 3,000g for 15 min at 4 °C, and plasma and red blood cells were separated and frozen at -20 °C within 1 h of collection. The frozen samples were transported on dry ice to the laboratory at the Institute of Reproductive and Child Health (Peking University, China) and stored at -70 °C until the assays were performed.
Measurements FRSs were calculated for all study participants. Plasma folate, total homocysteine, vitamin B12, and lipid concentrations, as well as blood pressure, were determined after 6 and 12 months of supplementation, and 6 months after supplementation cessation. Plasma folate concentrations were assessed using a microbial assay (Lactobacillus casei) [22], and plasma concentrations of total homocysteine and vitamin B12 were measured using an immunofluorescence method (AxSYM system, Abbott Laboratories, Missisauga, Canada). Plasma total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were measured enzymatically (Sekisui Medical Co., Ltd, Tokyo, Japan). Additionally, baseline hemoglobin concentrations were determined using a colorimetric method (Horiba ABX Micros 60, ABX Diagnostic, Montpellier, France). In the laboratory, the intra- and inter-assay coefficients of variation for the assays were \9 % for folate, \8 % for homocysteine, \10 % for vitamin B12, and \5 % for plasma lipid and hemoglobin. Blood pressure was measured using a mercury sphygmomanometer according to the recommendations of the British Hypertension Society [23]. The 10-year risk assessment for determining 10-year risk was determined according to the FRS. The FRS risk factors, which were derived from an update of the Framingham database and methodology, were calculated based on age, total plasma cholesterol, HDL cholesterol, systolic blood pressure, and current smoking status (smoker vs. non-smoker) by a specific prediction algorithm [19, 24]. First, the number of points for each risk factor was calculated. Then, the points for each risk factor were added to obtain the total risk score. The 10-year risk for myocardial infarction, angina, and coronary death was estimated from the total risk score. Sample size To estimate the sample size of the trial, a pilot study was conducted in the same county of the Hebei province in North China in 2007. The mean plasma homocysteine concentration from the pilot study was 18 lmol/L (SD 10.7). Based on data from previous studies, we hypothesized that a 30 % reduction in plasma homocysteine was associated with B vitamin supplementation in each of the six age- and gender-specific strata [25]. With a two-sided significance level of 0.05 and a b-error specification of 0.20, 50 participants were needed to detect a 30 % reduction in the treatment group. Assuming a worst-case scenario of a 20 % dropout rate, 60 participants were needed for each stratum; thus, a total of 360 participants were needed. The pilot study showed that the mean FRS was 9.3
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Eur J Nutr Fig. 1 Flow of study participants
405 eligible participants 9 refused to offer blood 6 refused to receive vitamin 390 Underwent randomization Months Start 0
195 Assigned to treatment group
Supplementation
1 died 5 moved 9 Dropped out 6
180 blood sample collected and assayed 1 died 2 moved 4 Dropped out
Stopping Supplementation
Stop 12
18
173 blood sample collected and assayed 2 died 2 moved 3 Dropped out 166 blood sample collected and assayed
(SD 5.3). With a two-sided significance level of 0.05 and a sample size of 360, we have 80 % power to find a difference of 15 % of FRS between the two groups. Statistical analysis Compliance was assessed by the number of supplements consumed divided by the number of days from enrollment to the end of the intervention. We examined the randomization effectiveness by comparing the key baseline characteristics between the two groups. Quantitative variables were ln-transformed to achieve an approximate normal distribution. The chi-square test or independent samples t test was used to examine statistical differences between the two groups. The paired-samples t test was used to analyze changes in folate and B12 concentrations from baseline to 6, 12, and 18 months. Hyperhomocysteinemia was defined as homocysteine concentrations C16.0 lmol/L [26], and folate deficiency was defined as plasma folate concentrations B6.8 nmol/L [27]. The effect of intervention on the FRS was assessed using a general linear model and Fisher’s least significant difference test. Subgroup analyses were performed using a general linear model with adjustment for baseline FRS, baseline homocysteine, baseline folate, age, gender, education, current smoking,
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195 Assigned to control group 3 died 6 Dropped out 186 blood sample collected and assayed 1 died 3 moved 8 Dropped out 174 blood sample collected and assayed 2 died 3 moved 6 Dropped out 163 blood sample collected and assayed
alcohol consumption, BMI, and MTHFR C677T. The correlation between B vitamin supplementation and baseline folate or homocysteine concentrations was tested by adding a product term to the model. p values less than 0.05 were considered statistically significant. Statistical analyses were performed using the Statistical Package for the Social Sciences version 18.0 (SPSS Inc., Chicago, IL, USA).
Results Among the 405 potential eligible participants, 390 underwent randomization, 366 supplemented their diet for 6 months, 347 supplemented their diet for 12 months, and 329 completed the entire study, including the 6-month follow-up period after supplementation cessation (Fig. 1). Demographic characteristics were similar between the subjects who dropped out and those who participated in the entire study. No adverse events related to supplementation were reported during the study period. Baseline characteristics between the two groups were balanced (Table 1). The mean age, BMI, plasma folate, and plasma homocysteine concentrations were 66.8 years (SD 4.4), 23.5 kg/m2 (SD 3.7), 9.6 nmol/L (95 % CI 9.1, 10.1), and 19.8 lmol/L (95 % CI 18.8, 20.8), respectively.
Eur J Nutr Table 1 Baseline characteristics of the study population: elderly Chinese individuals aged 60–74 years (n = 390) Characteristics
Treatment group (n = 195)
Control group (n = 195)
p value*
Age, years, mean (SD)
66.7 (4.5)
66.8 (4.2)
0.72
Male (%)
49.2
50.8
0.76
Han ethnicity (%)
100.0
99.0
0.16
Farmer (%)
95.9
99.0
0.06
Illiteracy (%)
40.0
38.5
0.76
Current smoking (%)
27.2
24.6
0.56
Alcohol consumption (%)
15.4
15.9
0.89
Body mass index (kg/m2) mean (SD)
23.6 (3.9)
23.4 (3.5)
0.66
Hb, g/L, mean (SD)
149.5 (18.6)
149.9 (14.8)
0.81
Systolic blood pressure, mmHg, mean (SD)
133.2 (17.9)
131.2 (16.9)
0.25
Diastolic blood pressure, mmHg, mean (SD) Plasma folate, nmol/L, geometric mean (95 % CI)
79.3 (8.5) 9.9 (9.2, 10.6)
77.8 (8.3) 9.3 (8.7, 10.1)
0.07 0.30
Plasma folate deficiency (%)a
25.1
24.6
0.91
Red blood cell folate, nmol/L, geometric mean (95 % CI)
467.4 (436.4, 500.5)
466.7 (437.9, 497.3)
0.97
Plasma homocysteine concentrations, lmol/L, mean (95 % CI)
20.0 (18.6, 21.6)
19.5 (18.2, 20.9)
0.60
Hyperhomocysteinemia (%)b
59.5
62.1
0.60
Vitamin B12, pg/mL, geometric mean (95 % CI)c
189.4 (148.2, 242.1)
232.4 (202.2, 267.1)
0.16
Vitamin B12 deficiency (%)c
46.0
38.0
0.42
Total cholesterol, mmol/L, mean (SD)
4.22 (1.02)
4.23 (1.17)
0.89
High-density lipoprotein cholesterol, mmol/L, mean (SD)
1.17 (0.33)
1.19 (0.35)
0.54
Low-density lipoprotein cholesterol, mmol/L, mean (SD)
2.29 (0.74)
2.26 (0.76)
0.73
Triglyceride, mmol/L, mean (SD)
0.97 (0.42)
1.05 (0.41)
0.09
Framingham risk score, mean (95 % CI)
9.26 (8.30, 10.22)
9.01 (8.07, 9.95)
MTHFR C677T variant (%) CC
0.71 0.55
12.4
11.2
CT
49.7
45.3
TT
37.9
43.6
* p value from t test for quantitative variables and v2 test for qualitative variables a
Plasma folate deficiency was defined as plasma folate concentration B6.8 nmol/L
b
Hyperhomocysteinemia was defined as plasma homocysteine concentration C16.0 lmol/L
c
Plasma vitamin B12 deficiency was defined as plasma vitamin B12 concentration\203 pg/mL. Fifty samples from each group were assayed for plasma vitamin B12 concentrations
MTHFR 677TT genotype carriers represented 40.7 % of the study population. Almost all participants were of Han ethnicity and were farmers. The average compliance after 6 and 12 months of supplementation was 97 % and 90 %, respectively (both of which did not differ between the two groups). Plasma folate and B12 concentrations after 6, 12, and 18 months of supplementation are shown in Fig. 2. Compared with the control, the folate concentration in the treatment group increased by 253 % after 6 months (treatment group 38.6 nmol/L vs. control group 10.9 nmol/L; p \ 0.001) and by 172 % after 12 months (treatment group 37.2 nmol/L vs. control group 13.7 nmol/L; p \ 0.001). Vitamin B12 plasma concentrations increased by 80 % after 6 months (371 vs. 206 pg/mL; p \ 0.001) and 60 % after 12 months (343 vs. 214 pg/mL; p \ 0.001). Continued supplementation for 12 months
resulted in no further increase in folate or vitamin B12 concentrations compared to concentrations achieved at 6 months. Significant increases in folate concentrations (30 %, 17.2 vs. 13.2 nmol/L; p \ 0.001) and in vitamin B12 concentrations (31 %, 291 vs. 222 pg/mL; p = 0.043) were observed at 18 months, 6 months after supplementation cessation. Mean baseline homocysteine concentration was 20.0 (95 % CI 18.6, 21.6) lmol/L in the treatment and 19.5 (95 % CI 18.2, 20.9) lmol/L in the control (t = 0.519, p = 0.60). Compared with the control, supplementation with B vitamins reduced plasma homocysteine by 35.6 % (12.9 vs. 20.1 lmol/L; p \ 0.001) at 6 months after supplementation and by 43.4 % (mean difference -10.0 lmol/ L; 95 % CI -12.0, -8.1 lmol/L; p \ 0.001) at 12 months after supplementation.
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Plasma folate b
50
Geometric mean
*** ‡
40
*** ‡
30 *** ‡
20 10 0
0
6
12
18
Time after randomization, months Fig. 2 Folate (a) and vitamin B12 (b) concentrations after randomization (supplementation stopped after 12 months). Error bars indicate 95 % confidence intervals. Values were ln-transformed before
Geometric mean
a
Vitamin B12 treatment
500
*** ‡
*** ‡
400
control *** †
300 200 100 0
0
6
12
18
Time after randomization, months analysis. Compared with baseline, paired-samples t test in treatment group, ***p \ 0.001. Compared with the control group, independent samples t test, àp \ 0.001, p \ 0.05
Table 2 Framingham risk score on coronary heart disease after randomization (supplementation stopped after 12 months) n Control
Differencea
Mean (95 % CI)* Treatment
%b
Control group
Treatment group
Mean (95 % CI)
p value
Framingham risk score (unadjusted) 6 months 12 months
183 169
179 170
10.17 (9.19, 11.14) 10.01 (8.97, 11.04)
10.10 (9.11, 11.08) 9.41 (8.38, 10.44)
-0.07 (-1.46, 1.31) -0.59 (-2.06, 0.87)
0.916 0.425
-0.7 -5.9
18 months
156
158
9.72 (8.68, 10.77)
9.49 (8.45, 10.54)
-0.23 (-1.71, 1.25)
0.759
-2.4
Framingham risk score (adjusted)c 6 months
183
179
10.31 (9.83, 10.80)
9.94 (9.45, 10.42)
-0.38 (-1.06, 0.31)
0.279
-3.7
12 months
169
170
10.10 (9.60, 10.59)
9.33 (8.83, 9.83)
-0.77 (-1.47, -0.06)
0.033
-7.6
18 months
156
158
9.62 (9.11, 10.14)
9.55 (9.04, 10.07)
-0.07 (-0.80, 0.66)
0.855
-0.7
* Compared with baseline, the FRS at 6, 12, and 18 months increased in both the control and treatment groups (p \ 0.05) a
Difference between the treatment and placebo groups was determined by a general linear model and the Fisher’s least significant difference test, 95 % CI is shown in parentheses
b
Percent value = 100 9 difference of mean between treatment and control/mean of control
c
Values are estimated marginal means adjusted for baseline FRS
Mean baseline FRS was 9.26 (95 % CI 8.30, 10.22) in the treatment group and 9.01 (95 % CI 8.07, 9.95) in the control group (p = 0.71) (Table 1). Stratified analysis showed no difference in baseline FRS between the treatment and control groups, as determined by evaluating folate and vitamin B12 concentrations. Compared with baseline concentrations, the FRS after 6, 12, and 18 months increased in both the control and treatment groups (p \ 0.05). Compared with the control group, there was no significant effect of B vitamin supplements on the FRS after 6 months (mean difference -0.38; 95 % CI -1.06, 0.31; p = 0.279), whereas a significant effect of supplementation was evident after 12 months (reduced magnitude 7.6 %; -0.77; 95 % CI -1.47, -0.06; p = 0.033), but this reduction disappeared 6 months after supplementation cessation (-0.07; 95 % CI -0.80, 0.66; p = 0.855) (Table 2). We further observed that the reduction effects existed in all strata classified by baseline folate or homocysteine
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concentration, and the magnitude of this reduction was more pronounced in people with baseline folate deficiency (2.9 % at 6 months; 10.4 % at 12 months) or hyperhomocysteinemia (2.8 % at 6 months; 7.5 % at 12 months) (Table 3). Similar subgroup results were also observed 6 months after the study participants stopped supplementing their diet. The main components of the FRS were examined after 6 and 12 months of supplementation, and 6 months after stopping supplementation (Table 4). Mean baseline HDL cholesterol was 1.17 (95 % CI 1.12, 1.22) in the treatment group and 1.19 (95 % CI 1.14, 1.24) in the control group (p = 0.542). Compared with the control, supplementation for 6 months showed a trend toward increasing HDL cholesterol by 3.4 % (0.04; 95 % CI -0.02, 0.10; p = 0.155), and supplementation for 12 months significantly increased HDL cholesterol by 9.2 % (0.11; 95 % CI 0.04, 0.18; p = 0.003); the magnitude of change decreased to 3.3 % (0.04; 95 % CI -0.02, 0.10; p = 0.194) 6 months
Eur J Nutr Table 3 Stratified analysis for FRS after randomization (supplementation stopped after 12 months) n
Differencea
Mean (95 % CI)*
Control
Treatment
%b
Control
Treatment
Mean (95 % CI)
p value
Baseline folate levelc B6.8 nmol/L 6 months
42
43
12.54 (11.38, 13.72)
12.19 (11.03, 13.34)
-0.36 (-2.08, 1.37)
0.682
-2.9
12 months
40
40
12.50 (11.66, 13.34)
11.20 (10.36, 12.04)
-1.30 (-2.54, -0.07)
0.039
-10.4
18 months
34
36
12.54 (11.38, 13.70)
11.60 (10.48, 12.72)
-0.94 (-2.62, 0.74)
0.265
-7.5
6 months 12 months
141 129
135 129
9.29 (8.79, 9.79) 9.16 (8.64, 9.68)
9.26 (8.75, 9.77) 8.78 (8.26,9.30)
-0.03 (-0.75, 0.69) -0.38 (-1.12, 0.36)
0.935 0.313
-0.3 -4.1
18 months
122
121
8.67 (8.11, 9.23)
8.95 (8.40, 9.50)
0.28 (-0.51, 1.06)
0.491
3.2
[6.8 nmol/L
Baseline homocysteine levelc \16.0 lmol/L 6 months
72
71
8.41 (7.75, 9.07)
8.33 (7.69, 8.98)
-0.07 (-1.01, 0.86)
0.875
-0.8
12 months
64
68
8.43 (7.73, 9.13)
7.82 (7.16, 8.47)
-0.61 (-1.59, 0.36)
0.214
-7.2
18 months
60
63
8.05 (7.30, 8.80)
7.91 (7.20, 8.61)
-0.14 (-1.19, 0.90)
0.786
-1.7
6 months
111
107
11.25 (10.61, 11.89)
10.93 (10.28, 11.59)
-0.32 (-1.25, 0.61)
0.497
-2.8
12 months
105
101
11.05 (10.49, 11.62)
10.23 (9.65, 10.81)
-0.83 (-1.64, -0.01)
0.048
-7.5
18 months
96
94
10.56 (9.88, 11.24)
10.57 (9.88, 11.27)
0.02 (-0.97, 1.00)
0.976
0.2
C16.0 lmol/L
* Values were estimated by marginal means adjusted for baseline FRS, baseline homocysteine level, baseline folate level, age, gender, education, current smoking, alcohol consumption, body mass index, and MTHFR C677T; 95 % CI is shown in parentheses a
Difference between the treatment and placebo groups was determined by a general linear model with adjustment for the variables listed above and by the Fisher’s least significant difference test
b
Percent value = 100 9 difference of mean between treatment and control/adjusted mean of control
c
No significant interaction was found between treatment and baseline folate levels or baseline homocysteine levels using a general linear model with adjustment for the variables listed above
Table 4 Main components of FRS on coronary heart disease after randomization (supplementation stopped after 12 months) n Control
Differencea
Mean (95 % CI)* Treatment
Control
Treatment
Mean (95 % CI)
%b p value
High-density lipoprotein cholesterol (mmol/L) 6 months
183
179
1.19 (1.14, 1.23)
1.23 (1.19, 1.27)
0.04 (-0.02, 0.10)
0.155
3.4
12 months
169
170
1.17 (1.12, 1.22)
1.27 (1.22, 1.32)
0.11 (0.04, 0.18)
0.003
9.2
18 months
156
158
1.20 (1.16, 1.24)
1.24 (1.20, 1.28)
0.04 (-0.02, 0.10)
0.194
3.3
Total plasma cholesterol (mmol/L) 6 months
183
179
4.48 (4.36, 4.61)
4.42 (4.29, 4.55)
-0.06 (-0.24, 0.12)
0.499
-1.3
12 months 18 months
169 156
170 158
4.01 (3.88, 4.14) 4.32 (4.20, 4.43)
4.01 (3.89, 4.14) 4.38 (4.27, 4.49)
0.00 (-0.17, 0.18) 0.06 (-0.10, 0.22)
0.960 0.452
0.0 0.0
Systolic blood pressure (mmHg) 6 months
183
179
140.6 (138.0, 143.3)
142.5 (139.8, 145.2)
1.86 (-1.91, 5.64)
0.332
1.3
12 months
169
170
134.5 (131.9, 137.0)
133.6 (131.0, 136.2)
-0.89 (-4.50, 2.72)
0.628
-0.7
18 months
156
158
139.8 (137.2, 142.4)
138.7 (136.2, 141.3)
-1.04 (-4.66, 2.58)
0.573
-0.7
* Values are estimated marginal means adjusted for baseline level of each component a
Difference between the treatment and placebo groups was determined using a general linear model and by the Fisher’s least significant difference test; 95 % CI is shown in parentheses b Percent value = 100 9 difference of mean between treatment and control/mean of control
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after stopping supplementation. No significant change was found in total plasma cholesterol or systolic blood pressure.
Discussion In this randomized controlled trial, we found that daily supplementation with a low dosage of folic acid, vitamin B6, and vitamin B12 for 12 months effectively reduced the FRS, particularly in healthy elderly people with a folate deficiency. We also found that B vitamins increased HDL cholesterol after 12 months. The magnitude of reduction in the FRS and HDL cholesterol returned to baseline after 6 months of supplementation cessation. Despite the fact that the FRS has been used as a primary outcome measure in trials to investigate the effects of nutraceutical supplements [28] and fruit and vegetable intake [29], to date, no trial has examined the effects of low-dose complex B vitamins on the FRS. Here, we found that 12-month supplementation with B vitamins reduced the FRS by 7.6 % overall and by 11 % in folate-deficient elderly people compared to the control group, although the FRS at 12 months increased in both the control and treatment groups with age compared with baseline. The reduction in magnitude of the FRS declined after supplementation cessation, further indicating a need for persistent supplementation to maintain the associated benefits. Additionally, the participants in our clinical trial did not encounter any serious adverse events related to the supplementation, indicating the feasibility of potential universal supplementation with B vitamins near the RDA in healthy elderly individuals. Although no trial has investigated the effects of lowdose B-complex vitamins on the FRS in healthy adults, some trials have investigated the effects of high-dose B vitamins on other functional (e.g., exercise electrocardiography testing) [30] or morphological indicators (e.g., carotid arterial stiffness and carotid intima-media thickness) of cardiovascular diseases [5]. Indeed, previous trials like the one described in this report have shown the beneficial effects on functional indicators [30], but not for morphological markers [5]. This is most likely due to a long imperceptible process in the development of morphological changes [31], which may further highlight the importance of prophylactic supplementation of B vitamins in the primary prevention of cardiovascular diseases. The mechanism underlying the effect of B vitamins on improving functional indicators is possibly related to their role in promoting the re-methylation of homocysteine [32], scavenging superoxide radicals [33], and/or advancing blood lipid metabolism [34]. We found that plasma HDL cholesterol significantly increased by 9.2 % 12 months after supplementation with B vitamins; however, this
123
elevation decreased after supplementation ceased. Similar patterns were observed between changes in FRS and plasma HDL cholesterol, indicating that advancing blood lipid metabolism might be one of the major mechanisms underlying the beneficial effect of B vitamins on cardiovascular diseases. Our findings are in accordance with previous studies, which demonstrated that folic acid supplementation in healthy postmenopausal Italian women increased HDL cholesterol levels by 6 and 12 % after 1 month and 8 weeks, respectively, of folic acid administration (7.5 mg/day) [34, 35]. Additionally, an observational study in young healthy non-smokers showed that plasma folate levels were associated with HDL cholesterol levels and endothelial function in the brachial artery, regardless of homocysteine status, indicating that low folate levels may be an independent risk factor for cardiovascular diseases [36]. The mechanism underlying the correlation between B vitamin supplementation and increased HDL cholesterol levels might be related to improvement of hepatic metabolism and antioxidation [34, 35]. Increased HDL cholesterol can protect endothelial function by stimulating reverse cholesterol transport, antioxidant, anti-apoptotic, and/or anti-inflammatory properties, all of which are beneficial for cardiovascular health [37]. In this randomized controlled trial, we also found that both folate and vitamin B12 concentrations in the treatment group increased and reached a high level after 6 months of supplementation, stopped increasing with continued supplementation for a total of 12 months, and returned to baseline after 6 months of supplementation cessation. A similar change in pattern was reported by Crider et al. [38], who demonstrated that the highest level of folate was achieved 3 months or 6 months after supplementation of folic acid at doses of 100, 400, and 4,000 lg/day and 4,000 lg/week in Northern Chinese women of childbearing age; a significant decrease was observed after 3 months of supplementation cessation in all dose groups, regardless of MTHFR genotype [38]. Therefore, it appears that there is a plateau for increased plasma folate and vitamin B12 level after 3 or 6 months of supplementation, and the intervention-enhanced folate status rapidly diminishes once supplementation ceases. Our trial had several strengths. First, the dosage of B vitamins was close to the RDA [39] and did not cause serious adverse effects. This is important, as an overdose of folic acid can promote cell proliferation, raise asymmetric dimethylarginine levels, and affect pro-atherogenic gene expression, which might induce unexpected toxicity [40, 41]. Second, the fortification of foods with folic acid was not available in our study area, and folate and vitamin B12 deficiency was prevalent in our study population, which provided us with the opportunity to examine the benefits of
Eur J Nutr
B vitamins at a low dosage. In several previous large-scale trials, folate fortification and high baseline folate levels in the study areas might have contributed to the negative results of the effects of B vitamins on the prevention of cardiovascular events [8–10]. Despite these strengths, it should be noted that the FRS has not been well validated in the Chinese population, which may affect the validity of our findings. However, the FRS is calculated based on several well-accepted classic risk factors for cardiovascular diseases, indicating that our findings are most likely valid. In conclusion, daily supplementation with low-dose B vitamins for 12 months reduced the FRS, especially in healthy elderly people with a folate deficiency. The benefits diminished after supplementation cessation, indicating a need for the persistent use of low-dose B vitamins to maintain the associated benefits. Future work is warranted to examine whether the results are applicable to people in other societies or in Chinese people less than 60 years of age.
9.
10.
11.
12.
13.
14.
15. Acknowledgments This study was supported by two grants from the National Natural Science Foundation of China (Grant Nos. 30572071; 30471486). We thank Dr. Zuguo Mei (U.S. Centers for Disease Control and Prevention) and Dr. Shufeng Zhou (University of South Florida) for their thoughtful review and editing of this manuscript. We thank all participants for their dedication and the local health workers for their assistance with subject recruitment and data collection.
16.
17. Conflict of interest
The authors declare no conflicts of interest.
References 18. 1. Ueland PM, Loscalzo J (2012) Homocysteine and cardiovascular risk: the perils of reductionism in a complex system. Clin Chem 58:1623–1625 2. McCully KS (2007) Homocysteine, vitamins, and vascular disease prevention. Am J Clin Nutr 86:1563S–1568S 3. Hustad S, Midttun O, Schneede J, Vollset SE, Grotmol T, Ueland PM (2007) The methylenetetrahydrofolate reductase 677C?T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism. Am J Hum Genet 80:846–855 4. Robinson K (2004) Renal disease, homocysteine, and cardiovascular complications. Circulation 109:294–295 5. Durga J, Bots ML, Schouten EG, Grobbee DE, Kok FJ, Verhoef P (2011) Effect of 3 y of folic acid supplementation on the progression of carotid intima-media thickness and carotid arterial stiffness in older adults. Am J Clin Nutr 93:941–949 6. Armitage JM, Bowman L, Clarke RJ, Wallendszus K, Bulbulia R, Rahimi K, Haynes R, Parish S, Sleight P, Peto R, Collins R (2010) Effects of homocysteine-lowering with folic acid plus vitamin B12 vs. placebo on mortality and major morbidity in myocardial infarction survivors: a randomized trial. JAMA 303:2486–2494 7. Clarke R, Halsey J, Bennett D, Lewington S (2011) Homocysteine and vascular disease: review of published results of the homocysteine-lowering trials. J Inherit Metab Dis 34:83–91 8. Bonaa KH, Njolstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, Wang H, Nordrehaug JE, Arnesen E, Rasmussen K
19.
20.
21.
22.
23.
24.
(2006) Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 354:1578–1588 Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, McQueen MJ, Probstfield J, Fodor G, Held C, Genest J Jr (2006) Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 354:1567–1577 The VITATOPS Trial Study Group (2010) B vitamins in patients with recent transient ischaemic attack or stroke in the VITAmins TO Prevent Stroke (VITATOPS) trial: a randomised, doubleblind, parallel, placebo-controlled trial. Lancet Neurol 9:855–865 Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ (1998) Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279:359–364 Voutilainen S, Rissanen TH, Virtanen J, Lakka TA, Salonen JT (2001) Low dietary folate intake is associated with an excess incidence of acute coronary events: the Kuopio Ischemic Heart Disease Risk Factor Study. Circulation 103:2674–2680 Lee M, Hong KS, Chang SC, Saver JL (2010) Efficacy of homocysteine-lowering therapy with folic acid in stroke prevention: a meta-analysis. Stroke 41:1205–1212 Wang X, Qin X, Demirtas H, Li J, Mao G, Huo Y, Sun N, Liu L, Xu X (2007) Efficacy of folic acid supplementation in stroke prevention: a meta-analysis. Lancet 369:1876–1882 US Food and Drug Administration (2000) Letter regarding dietary supplement health claim for folic acid, vitamin B6, and vitamin B12 and vascular disease (Docket No. 99P-3029). http:// www.fda.gov/Food/IngredientsPackagingLabeling/LabelingNutri tion/ucm072865.htm#tab US Food and Drug Administration (2001) Settlement reached for health claim relating B vitamins and vascular disease. http:// www.fda.gov/Food/IngredientsPackagingLabeling/LabelingNutri tion/ucm072855.htm Elshorbagy AK, Oulhaj A, Konstantinova S, Nurk E, Ueland PM, Tell GS, Nygard O, Vollset SE, Refsum H (2007) Plasma creatinine as a determinant of plasma total homocysteine concentrations in the Hordaland Homocysteine Study: use of statistical modeling to determine reference limits. Clin Biochem 40:1209–1218 Gale CR, Ashurst H, Phillips NJ, Moat SJ, Bonham JR, Martyn CN (2001) Renal function, plasma homocysteine and carotid atherosclerosis in elderly people. Atherosclerosis 154:141–146 National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (2002) Third report of the National Cholesterol Education Program (NCEP) expert panel on detection E, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 106:3143–3421 House AA, Eliasziw M, Cattran DC, Churchill DN, Oliver MJ, Fine A, Dresser GK, Spence JD (2010) Effect of B-vitamin therapy on progression of diabetic nephropathy: a randomized controlled trial. JAMA 303:1603–1609 Tighe P, Ward M, McNulty H, Finnegan O, Dunne A, Strain J, Molloy AM, Duffy M, Pentieva K, Scott JM (2011) A dosefinding trial of the effect of long-term folic acid intervention: implications for food fortification policy. Am J Clin Nutr 93:11–18 O’Broin S, Kelleher B (1992) Microbiological assay on microtitre plates of folate in serum and red cells. J Clin Pathol 45:344–347 Petrie JC, O’Brien ET, Littler WA, de Swiet M (1986) Recommendations on blood pressure measurement. Br Med J (Clin Res Ed) 293:611–615 Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB (1998) Prediction of coronary heart disease using risk factor categories. Circulation 97:1837–1847
123
Eur J Nutr 25. Collaboration Homocysteine Lowering Trialists’ (2005) Dosedependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials. Am J Clin Nutr 82:806–812 26. Ueland PM, Refsum H, Stabler SP, Malinow MR, Andersson A, Allen RH (1993) Total homocysteine in plasma or serum: methods and clinical applications. Clin Chem 39:1764–1779 27. Federation of American Societies for Experimental Biology (1984) Assessment of the folate nutritional status of the U.S. population based on data collected in the second National Health and Nutrition Examination Survey, 1976–1980. Federation of American Societies for Experimental Biology, Bethesda, MD 28. Izzo R, de Simone G, Giudice R, Chinali M, Trimarco V, De Luca N, Trimarco B (2010) Effects of nutraceuticals on prevalence of metabolic syndrome and on calculated Framingham Risk Score in individuals with dyslipidemia. J Hypertens 28:1482–1487 29. Bhupathiraju SN, Tucker KL (2011) Greater variety in fruit and vegetable intake is associated with lower inflammation in Puerto Rican adults. Am J Clin Nutr 93:37–46 30. Vermeulen EG, Stehouwer CD, Twisk JW, van den Berg M, de Jong SC, Mackaay AJ, van Campen CM, Visser FC, Jakobs CA, Bulterjis EJ, Rauwerda JA (2000) Effect of homocysteine-lowering treatment with folic acid plus vitamin B6 on progression of subclinical atherosclerosis: a randomised, placebo-controlled trial. Lancet 355:517–522 31. Morrison KM, Atkinson SA, Yusuf S, Bourgeois J, McDonald S, McQueen MJ, Persadie R, Hunter B, Pogue J, Teo K (2009) The Family Atherosclerosis Monitoring In early life (FAMILY) study: rationale, design, and baseline data of a study examining the early determinants of atherosclerosis. Am Heart J 158:533–539 32. Wilcken DE, Dudman NP, Tyrrell PA, Robertson MR (1988) Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: possible implications for prevention of vascular disease. Metabolism 37:697–701
123
33. Verhaar MC, Stroes E, Rabelink TJ (2002) Folates and cardiovascular disease. Arterioscler Thromb Vasc Biol 22:6–13 34. Villa P, Perri C, Suriano R, Cucinelli F, Panunzi S, Ranieri M, Mele C, Lanzone A (2005) L-folic acid supplementation in healthy postmenopausal women: effect on homocysteine and glycolipid metabolism. J Clin Endocrinol Metab 90:4622–4629 35. Paradisi G, Cucinelli F, Mele MC, Barini A, Lanzone A, Caruso A (2004) Endothelial function in post-menopausal women: effect of folic acid supplementation. Hum Reprod 19:1031–1035 36. Imamura A, Murakami R, Takahashi R, Cheng XW, Numaguchi Y, Murohara T, Okumura K (2010) Low folate levels may be an atherogenic factor regardless of homocysteine levels in young healthy nonsmokers. Metabolism 59:728–733 37. Tran-Dinh A, Diallo D, Delbosc S, Varela-Perez LM, Dang QB, Lapergue B, Burillo E, Michel JB, Levoye A, Martin-Ventura JL, Meilhac O (2013) HDL and endothelial protection. Br J Pharmacol 169:493–511 38. Crider KS, Zhu JH, Hao L, Yang QH, Yang TP, Gindler J, Maneval DR, Quinlivan EP, Li Z, Bailey LB, Berry RJ (2011) MTHFR 677C?T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation. Am J Clin Nutr 93:1365–1372 39. Office of Dietary Supplements National Institutes of Health (2013) Dietary supplement fact sheet. http://ods.od.nih.gov/fact sheets/list-all/ 40. Antoniades C, Antonopoulos AS, Tousoulis D, Marinou K, Stefanadis C (2009) Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials. Eur Heart J 30:6–15 41. Loscalzo J (2006) Homocysteine trials—clear outcomes for complex reasons. N Engl J Med 354:1629–1632
