Lowering Homocysteine with B Vitamins Has No Effect on Blood Pressure in Older Adults 1,2

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1 The Journal of Nutrition Nutrition and Disease Lowering Homocysteine with B Vitamins Has No Effect on Blood Pressure in Older Adults 1,2 Jennifer A. McMahon, 3 C. Murray Skeaff, 3 Sheila M. Williams, 4 and Timothy J. Green 3 * 3 Department of Human Nutrition and 4 Department of Social and Preventive Medicine, University of Otago, Dunedin 9001, New Zealand Abstract An elevated circulating homocysteine concentration is associated with the risk of cardiovascular disease. The mechanism by which an elevated homocysteine increases cardiovascular risk is unclear but may be mediated in part by elevating blood pressure. It is well established that supplements containing folate, vitamins B-12, and B-6 lower homocysteine concentrations. However, the effect of homocysteine-lowering vitamins on blood pressure has not been well studied. We sought to determine whether lowering homocysteine with B vitamins lowers blood pressure in healthy older people with elevated homocysteine concentrations. Two hundred seventy-six healthy older participants ($65 y) with a homocysteine $13 mmol/l were randomized to receive a daily supplement containing folate (1 mg), vitamin B-12 (500 mg), and vitamin B-6 (10 mg), or a placebo, for 2 y. Plasma homocysteine was lower in the Vitamins group than the Placebo group at both 1[24.3 mmol/l (95% CI; 24.9, 23.7)] and 2 y [24.4 mmol/l (95% CI: 25.3, 23.6)]. Systolic and diastolic blood pressures as well as pulse pressure in the Vitamins group did not differ from the Placebo group over the duration of the trial. The mean differences in blood pressures, adjusted for baseline values, did not exceed 1 mm Hg. Supplemental B-vitamins lowered plasma homocysteine but had no effect on blood pressure in older people with elevated baseline homocysteine concentrations. J. Nutr. 137: , Introduction 1 Sponsored by an Otago Research Grant. Eprova (Switzerland) provided the supplements. 2 Author disclosure: J. A. McMahon, no conflicts of interest; C. M. Skeaff, no conflicts of interest; S. M. Williams, no conflicts of interest; and T. J. Green, no conflicts of interest. * To whom correspondence should be addressed. tim.green@stonebow. otago.ac.nz. An elevated circulating concentration of homocysteine is associated with an increased risk of coronary, cerebral, and peripheral vascular disease (1,2). The mechanism by which an elevated homocysteine increases cardiovascular risk is unclear but may be mediated in part by elevating blood pressure (3 5). Homocysteine may elevate blood pressure by damaging vascular endothelial integrity (6 9), increasing arterial stiffness (7,10,11), and reducing vasodilatory capacity (8,12). In animal (9) and in vitro studies (6), homocysteine administration caused direct endothelial cell injury. In humans, endothelium-dependent vasodilatation was impaired in chronic hyperhomocysteinemia (13) and acutely following a postmethionine load (14). Circulating homocysteine concentrations have been positively associated with blood pressure or hypertension in some (15 18) but not all (19,20) cross-sectional studies. In the Third NHANES ( ), serum homocysteine was independently associated with blood pressure, i.e., a ;5 mmol/l increase in homocysteine was associated with increases in diastolic and systolic blood pressure of 0.5 and 0.7 mm Hg in men and of 0.7 and 1.2 mm Hg in women (n ¼ 5978), respectively (17). Homocysteine may be lowered by taking supplements containing folic acid with or without vitamins B-12 or B-6 (21). Indeed, homocysteine-lowering vitamins have been shown to lower blood pressure in 2 small intervention trials (3,4). However, in the much larger Vitamin Intervention for Stroke Prevention (VISP) trial (n ¼ 1827) homocysteine reduction with 2.5 mg folic acid, 25 mg of pyridoxine, and 0.4 mg of vitamin B-12 had no effect on blood pressure relative to a control group (22). However, the participants in this study had a prior history of stroke; the majority of them, almost two-thirds, had a history of hypertension and were presumably taking antihypertensive medication. Also, the homocysteine reduction achieved in this study in the treated group was modest at 2 mmol/l. We conducted a 2-y randomized control trial to determine whether lowering homocysteine with folate, vitamin B-12, and vitamin B-6 reduces blood pressure in healthy older people with elevated homocysteine concentrations. Subjects and Methods The blood pressure results we report herein were a secondary endpoint of a 2-y trial to determine whether B-vitamins improved cognitive function in older people. The cognitive performance results are reported elsewhere (23). Participants. Participants $65 y of age were recruited from local service clubs (e.g., Rotary International), by advertising in newspapers, and by direct mail-outs. Participants were excluded if they were taking medications known to interfere with folate metabolism, their physician had told them they had probable dementia, were taking vitamin supplements containing folic acid, vitamin B-12, or vitamin B-6, were being /07 $8.00 ª 2007 American Society for Nutrition Manuscript received 16 October Initial review completed 11 November Revision accepted 14 February 2007.

2 treated for or had a history of depression, stroke, transient ischemic attacks, or diabetes. Volunteers were screened for plasma homocysteine and creatinine; those with a fasting homocysteine concentration,13 mmol/l, or plasma creatinine.133 mmol/l for men and.115 mmol/l for women, were excluded. Written informed consent was obtained from each participant prior to screening and the University of Otago Human Ethics Committee approved the study. Study design. Randomization of participants to treatment groups was done after all eligible participants had been identified through screening. Eligible participants were stratified using the median values of age and homocysteine concentration from the screening population (24). Thus, 4 strata were created: 1) those above both the median homocsyteine concentration and age, 2) those above the median homocysteine concentration and below the median age, 3) those below median homocysteine concentration and above the median age and, 4) those below both the median homocsyteine concentration and age. A random decimal between 0 and 1 was generated for each person in each of the 4 strata. Those with a random decimal below the median of the random decimal in each stratum were assigned to one group and the remainder to the other group. Participants were asked to consume 1 capsule daily for 2 y. Participants were provided with a 6-mo supply of capsules and asked to return every 6 mo to obtain a new supply. Participants returned unused capsules every 6 mo and these were counted to assess compliance. Participants could contact the study coordinator by phone. To further encourage compliance, nutrition newsletters were sent to the participants regularly during the study period. Supplements. The treatment capsules contained the microcrystalline cellulose as a filler plus 1000 mg folate (L-5-methyltetrahydrofolate, calcium salt), 500 mg vitamin B-12 (cyanocobalamin), and 10 mg vitamin B-6 (pyridoxine). The placebo capsules contained a blend of magnesium stearate and the filler (Merck Eprova). The investigators and the participants did not know the contents of the supplements. Blood pressure, blood collection, and laboratory methods. Blood pressure was measured at baseline, 1 y, and 2 y during cognitive testing using a Hawksley Random-Zero Sphygmomanometer (Hawksley and Sons). At each time point, blood pressure was recorded 20 min after the beginning of the cognitive assessment and then repeated 10 min later. The blood pressure was recorded in the sitting position and was recorded on the left arm. A third blood pressure recording was taken if there was.10 mm Hg difference in either the systolic or diastolic measures. The mean of the closest 2 blood pressure recordings was used in the analysis. With very few exceptions, blood pressure at baseline, 1 y, and 2y was recorded on the same day of the week and at the same time of day for each individual to minimize intrasubject variation. An overnight fasting blood sample was collected at baseline and every 6 mo thereafter. Plasma and serum were obtained by centrifuging the whole blood at g, for 15 min at 4 C within 2 h of collection. Blood samples were stored at 80 C until analyzed. Total homocysteine was measured using the IMx (Abbott Laboratories), Fluorescence Polarization Immunoassay (FPIA), measuring total L-homocysteine in plasma. Plasma folate concentrations were determined using a microbiological method on 96-well microplates, exactly as described by O Broin and Kelleher with chloramphenicol resistant Lactobacillus casei as the test microorganism (25). Plasma vitamin B-12 was measured using the ADVIA Centaur vitamin B-12 assay, a competitive immunoassay using direct chemiluminescent technology. Plasma creatinine was measured colorimetrically using Roche diagnostic kits on a Cobas Mira Analyzer (Roche Diagnostics). CV for these assays were 6.7% for plasma homocysteine, 7.7% for plasma folate, 5.6% for plasma vitamin B-12, and 7.2% for plasma creatinine. Likewise, multiple regression analyses, adjusting for baseline blood pressure, plasma creatinine, weight, height, and sex, were used to estimate the differences in blood pressure (systolic, diastolic, and pulse pressure). Results were considered significant at P, The statistical analysis for blood pressure was initially conducted on all participants (intent-to-treat) and then separately to test if there were any treatment differences between participants who were or were not taking antihypertensive medication. This was accomplished by including an interaction term for drug use by treatment group. Participants were considered antihypertensive medication users if they were taking one or more of either diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, or beta-adrenergic antagonists at the baseline visit. There was no interaction between type of treatment (Vitamins or Placebo) and time (y 1 or y 2) in any of the analyses, therefore, the differences between Vitamins and Placebo groups were based on the mean of 1- and 2-y blood pressures. All analyses were undertaken using STATA 9.0 software for Macintosh (Stata). Results Of 465 screened participants, we excluded 172 because they had a fasting plasma homocysteine,13 mmol/l and 3 because of an elevated plasma creatinine (Fig. 1). Two hundred seventy-six participants were randomized to the Placebo or Vitamins group. Before baseline measures were collected, 3 participants withdrew, leaving 273 participants. Twenty participants were lost to follow-up, 11 in the Placebo group and 9 in the Vitamins group. Fifteen participants discontinued taking the supplements but completed the study. Blood pressure recordings were available for 252 participants at baseline, 247 participants at y 1, and 249 participants at y 2. Overall, 214 of 252 participants (85%), for whom there were baseline blood pressure recordings, took at least 95% of their study capsules. Statistical analyses. Differences in characteristics of participants in the Vitamins and Placebo groups at baseline were determined using a chisquare test for categorical variables and Student s t test for continuous variables. Multiple regression analyses were used to estimate the differences in plasma homocysteine and folate after adjustment for baseline values at 1 and 2 y between the placebo group and the treatment group McMahon et al. FIGURE 1 Participant flow and follow-up.

3 Mean age, alcohol consumption, and antihypertensive medication use was similar between the groups at baseline (Table 1). Less than 5% of participants in either group smoked. Baseline vitamin and homocysteine concentrations were similar in both groups. A greater percentage of participants were women in the Placebo than the Vitamins group (52 vs. 37%; P ¼ 0.02). Plasma homocysteine was lower in the Vitamins group than the Placebo group at both 1 y [24.3 (95%CI; 24.9, 23.7) mmol/l] and 2 y [24.4 mmol/l (25.3, 23.6) mmol/l] (Fig. 2). Plasma folate concentrations concentrations were significantly higher in the Vitamins than the Placebo group at both 1 y [51 (48, 55) nmol/l] and 2 y [50 (46, 55) nmol/l]. Likewise, plasma vitamin B-12 concentrations were significantly higher in the Vitamins than the Placebo group at both 1 y [289 (258, 320) pmol/l] and 2 y [315 (278, 352) pmol/l]. Systolic and diastolic blood pressures as well as pulse pressure in the Vitamins group did not differ from the Placebo group over the duration of the trial (Table 2). The mean differences in blood pressures, adjusted for baseline values, did not exceed 1 mm Hg. Statistical adjustment of the regression analysis for known confounders of blood pressure altered these results little. Furthermore, controlling for weight change or including BMI rather than weight and height in the statistical model did not alter the results. Taking B vitamins had no effect on blood pressure in those who did or did not use antihypertensive medication. Among participants using antihypertensive medication, systolic blood pressure in the Vitamins group was 1.1 (23.6, 5.8) mm Hg higher than in the Placebo group. In those not using antihypertensive medication, systolic blood pressure was 0.3 (23.8, 4.3) mm Hg higher. In the statistical analysis for the interaction between treatment and antihypertensive medication use, the difference in systolic blood pressure between Vitamins and Placebo groups in medication users compared with nonusers was 0.8 (25.4, 7.0) mm Hg. For diastolic blood pressure, the treatment difference in medication users compared with nonusers was 2.0 (21.6, 5.7) mm Hg; for pulse pressure it was 21.1 TABLE 1 Baseline characteristic Baseline characteristics of study participants in each treatment group 1 Placebo, n ¼ 126 Vitamins, n ¼ 126 P-value Age at screening, y Women, n(%) 65 (52) 47 (37) 0.02 Current smoker, n(%) 1 (1) 5 (4) 0.25 BMI, kg/m Using antihypertensive 51 (40) 55 (44) 0.68 medication, n(%) High-normal hypertension or 39 (30) 32 (25) 0.42 hypertension, 2 n(%) Alcohol drinks, 3 drinks/wk MTHFR C677T TT, 4 n(%) 11 (9) 20 (16) 0.23 Plasma creatinine, mmol/l Plasma homocysteine, mmol/l Plasma folate, nmol/l Plasma vitamin B-12, pmol/l Values are means 6 SD or n (%). 2 Systolic blood pressure.130 mm Hg and diastolic blood pressure.80 mm Hg. 3 One drink was defined as 15 ml of pure alcohol; 360 ml beer, 150 ml wine, or 45 ml distilled spirits. 4 Methylene tetrahydrofolate reductase C677T, homozygous TT genotype. FIGURE 2 Plasma homocysteine (A), folate (B), and vitamin B-12 (C), concentrations in older people administered B vitamins or placebo for 2 y. Points are means 6 95% CI, n ¼ *Different from placebo, after adjusting for baseline values, P, (26.3, 4.1) mm Hg. The effect of B vitamins on blood pressure did not differ between males and females; the interaction between B vitamins and sex was not significant. Discussion To our knowledge, this is the first long-term study of healthy older people to examine the effect of homocysteine-lowering vitamins on blood pressure. In this randomized double-blind trial, B-vitamin supplementation for.2 y in older adults, with elevated baseline homocysteine concentrations ($13 mmol/l), lowered homocysteine concentrations by.25% relative to placebo but had no effect on either systolic or diastolic blood pressure. Our findings are consistent with those of the VISP where high-dose B-vitamin supplementation had no effect on systolic (140.2 vs mm Hg) or diastolic (77.3 vs mm Hg) blood pressure compared with low-dose supplementation Homocysteine-lowering vitamins and blood pressure 1185

4 TABLE 2 Blood pressure in older adults administered B vitamins or placebo for 2 y Baseline 1 Y1 Y2 Difference adjusted for Fully adjusted Blood pressure Placebo Vitamins Placebo Vitamins Placebo Vitamins baseline value 2 P-value difference 3 P-value All participants mm Hg n Systolic (20.3) (20.8) (17.5) (16.1) (18.7) (18.1) 0.5 (22.6, 3.5) (22.4, 3.7) 0.69 Diastolic 75.8 (11.6) 74.8 (13.2) 74.5 (10.5) 74.5 (11.1) 71.4 (10.1) 72.0 (11.9) 0.8 (21.0, 2.6) (20.9, 2.7) 0.35 Pulse pressure 62.2 (16.3) 61.8 (15.6) 62.3 (14.1) 61.5 (14.0) 62.6 (15.3) 62.6 (15.1) 20.2 (22.8, 2.3) (22.8, 2.4) 0.91 Antihypertensive medication users n Systolic (17.0) (20.5) (17.4) (17.5) (19.3) (19.3) 0.8 (23.9, 5.5) (23.6, 5.8) 0.64 Diastolic 76.0 (10.9) 74.1 (12.7) 72.8 (9.3) 75.4 (12.3) 71.9 (9.5) 71.3 (11.9) 1.9 (20.8, 4.7) (20.7, 4.8) 0.15 Pulse pressure 62.7 (15.1) 63.5 (15.0) 62.7 (16.2) 61.2 (14.0) 64.7 (16.1) 64.3 (16.3) 21.0 (25.0, 3.0) (24.7, 3.2) 0.71 Non-antihypertensive medication users n Systolic (22.4) (21.1) (17.6) (15.0) (18.3) (17.1) 0.2 (23.8, 4.2) (23.8, 4.3) 0.89 Diastolic 75.7 (12.1) 75.4 (13.5) 75.6 (11.2) 73.8 (10.1) 71.1 (10.5) 72.5 (11.9) 0.0 (22.4, 2.3) (22.4, 2.4) 0.99 Pulse pressure 61.9 (17.1) 60.5 (16.0) 62.1 (12.6) 61.8 (14.1) 61.2 (14.7) 61.2 (14.2) 0.3 (23.1, 3.7) (23.1, 3.7) Values are means 6 SD. The groups did not differ at baseline (Student s t-test). 2 Mean difference (95% CI) is based on an overall estimate of 1- and 2-y blood pressures. 3 Mean difference (95% CI). Regression analysis adjusted for baseline values, baseline body weight, height, plasma creatinine, and sex. over 2 y in 3361 subjects with a prior history of stroke (22). However, the mean baseline homocysteine was lower (13.4 vs mmol/l) and the homocysteine reduction achieved less (2 and 4 mmol/l) in the VISP trial than in our study. Our findings contrast to 2 smaller randomized trials in younger people with no history of hypertension at baseline (3,4). In the first study, 24 long-term smokers were randomized to folic acid or placebo for 4 wk (4). Mean systolic and diastolic blood pressure dropped 8 and 4 mm Hg, respectively, in the folic acid group with no change in the placebo group. In a second study, of 130 participants with an elevated baseline postmethinonine load test, taking folic acid and vitamin B-6 for up to 2 y lowered homocysteine by.7 mmol/l and lowered systolic and diastolic blood pressure by 3.7 and 1.9 mm Hg, respectively (3). It is unclear why our findings differ from these earlier trials. Our trial participants were older and had a higher mean baseline systolic and diastolic blood pressure than participants in these trials. The supplement in our trial consisted of 1 mg of folate, 500 mg vitamin B-12, and 10 mg vitamin B-6, whereas a supplement containing 5 mg of folic acid was given in the smokers study, and 5 mg of folic acid and 250 mg of vitamin B-6 was given in the other study. Although similar reductions in homocysteine were reported in the other studies and ours, perhaps higher doses of these vitamins are required to lower blood pressure. Finally, in contrast to the other studies, our study included few current smokers. Smoking has been associated with endothelial dysfunction and arterial stiffening (26,27); it is possible that the response to B vitamins is different in smokers compared with nonsmokers. Noncompliance probably does not explain our null findings, as the self-reported high compliance is supported by lower homocysteine and substantially higher plasma folate concentration in the Vitamins group. Our study did include a high proportion of antihypertensive medication users, which might have obscured a blood pressure lowering effect of B vitamins. However, a subanalysis that excluded individuals taking antihypertensive medication did not show an effect of B vitamins on blood pressure. The majority of participants in our trial did not have hypertension so perhaps it is not unexpected that B vitamins did not lower blood pressure in this group. However, dietary intervention has lowered blood pressure in studies of people with similar baseline blood pressure. For example, in the Dietary Approaches to Stop Hypertension (DASH) trial, baseline systolic and diastolic blood pressures were (mean 6 SD) and mm Hg, respectively, compared with and mm Hg in our study (28). The DASH diet reduced systolic and diastolic blood pressure by 6.7 and 3.0 mm Hg, respectively. Further, in a subanalysis that was restricted to individuals with baseline systolic and diastolic blood pressure of,140 and 90 mm Hg, respectively, blood pressure was reduced in those who followed the DASH diet, but less so than those with hypertension. An analysis including only individuals with a baseline systolic blood pressure.130 mm Hg and a diastolic blood pressure.80 mm Hg (n ¼ 72) did not reveal a blood pressure lowering effect of B vitamins (data not shown). In a recent prospective study from the Framingham Cohort, there was no association between baseline homocysteine and subsequent risk of hypertension (19). However, in the Nurses Health Study, women who consumed.1000 mg/d folic acid had a lower relative risk [0.54 (95% CI, )] of incident selfreported hypertension over 8 y of follow-up (29). It is possible that additional folate may lower blood pressure independently of its effect on homocysteine. Indeed, folic acid has been shown to acutely improve endothelial function before a decline in homocysteine. In the small study of smokers reported above, changes in plasma folate, not homocysteine, predicted changes in systolic blood pressure (4). It is possible that individuals prescreened for low folate status might have a different response. However, in the present study, we could not disentangle the treatment of increasing folate status and decreasing homocysteine. In conclusion, B vitamins substantially lowered plasma homocysteine but had no effect on blood pressure in older people with elevated baseline homocysteine concentrations. If the relation between vascular disease and homocysteine is 1186 McMahon et al.

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