Effects of multivitamins and low-dose folic acid supplements on flow-mediated vasodilation and plasma homocysteine levels in older adults☆

Article Outline

• Abstract
• Methods
  • Subjects
  • Study design
  • Laboratory measurements
  • Brachial artery reactivity
  • Statistical analysis
• Results
  • Baseline subject characteristics
  • Laboratory results
  • Brachial artery reactivity results
  • Adverse events
• Discussion
  • Limitations
  • Conclusions
• References
• Copyright

Abstract 

Background

Hyperhomocysteinemia is associated with aging, endothelial dysfunction, and increased risk of coronary heart disease in older adults; however, the effects of homocysteine-lowering therapy on vascular reactivity in older persons are unknown. The objective of this study was to determine the effects of multivitamins (MV) and folic acid (FA) supplements on flow-mediated vasodilation (FMD) in older adults.

Methods

Individuals ≥70 years old with homocysteine levels ≥10 μmol/L were recruited for this 40-week, prospective, single-blinded study. All subjects were treated sequentially, with each of the following daily therapies for 10 weeks: (1) placebo, (2) MV (400 μg FA, 6 mg vitamin B₆, 25 μg vitamin B₁₂), (3) placebo, then (4) MV + FA (total FA, 1400 μg). FMD, folate intake, and laboratory values were measured at each visit. Investigators were blinded to subject treatment phase when measuring vessel diameters and calculating FMD.

Results

Twenty subjects (mean ± SEM age, 78.0 ± 1.2 [range, 70 to 88] years, 9 women) completed the MV and 17 completed the MV + FA interventions. FMD was impaired at baseline (2.0% ± 1.2%). During the 40-week study, homocysteine levels decreased by 1.4 ± 0.9 μmol/L (p_trend = 0.034) from a baseline of 12.8 ± 0.6 μmol/L; however, FMD did not change significantly (p_trend = 0.874). FMD did not improve after therapy with MV alone (3.0% ± 0.9% [week 10] vs 2.4% ± 1.1% [week 20], P = .716) or with MV + FA (2.6% ± 0.9% [week 30] vs 1.9% ± 0.7% [week 40], P = .484).

Conclusions

At doses commonly prescribed in clinical practice, MV and FA supplements did not improve FMD in older adults with hyperhomocysteinemia.

Aging is associated with endothelial dysfunction, arterial thickening, and impaired arterial compliance.¹, ², ³ These components of “age-related” vascular damage may be mediated in part by increased exposure time to age-dependent risk factors.³ With aging, the prevalence of elevated plasma homocysteine (Hcy) levels increases, a finding associated with an increased risk of coronary heart disease (CHD) in older adults.⁴, ⁵, ⁶ Hyperhomocysteinemia is associated with impaired endothelial function and vascular smooth muscle cell proliferation — two key components of “age-related” vascular damage.⁷, ⁸, ⁹ It is unclear, however, if the cumulative adverse effects of Hcy on the endothelium and vascular smooth muscle cells in older persons are reversible with Hcy-lowering therapy.

Although folic acid (FA) and B-vitamin supplements reduce Hcy levels in older adults, their effects on vascular function in older persons are unknown.¹⁰, ¹¹, ¹², ¹³ In young and middle-aged adults, Hcy-lowering therapy improves endothelium-dependent vasodilation, a predictor of future cardiovascular events.¹⁴, ¹⁵, ¹⁶, ¹⁷, ¹⁸, ¹⁹, ²⁰, ²¹ The effects of Hcy lowering on endothelial function in individuals over the age of 70 years, the segment of the population most at risk for CHD and its complications, are not known. Since the proliferation of vascular smooth muscle cells and decreased arterial compliance seen in older adults theoretically may lead to impaired vasodilator reserve, it is unclear whether geriatric patients will have the same improvement in endothelial function as observed in young and middle-aged adults.

The purpose of this study was to evaluate the effects of FA and B-vitamin supplementation on flow-mediated vasodilation (FMD) of the brachial artery in older adults with hyperhomocysteinemia. Given the uncertainty regarding the effectiveness and optimal treatment dose for Hcy reduction in older patients, a secondary end point of this study was to evaluate the effects of folic acid and B-vitamin supplements, at doses commonly used in clinical practice, on plasma Hcy levels.¹², ²², ²³

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Methods 

The Institutional Review Board of the University of Wisconsin Medical School approved this study. All subjects provided informed consent before participation.

Subjects 

Individuals ≥70 years old with Hcy levels ≥10 μmol/L were recruited for this 40-week, prospective, sequential therapy, single-blinded study. Older adults being followed in outpatient geriatrics and cardiovascular medicine clinics were invited to participate by mass mailings and by individual contact with their physician. Subjects were excluded if they currently used tobacco or consumed >3 alcoholic beverages daily, had untreated secondary causes of hyperhomocysteinemia (such as vitamin B₁₂ deficiency), chronic renal insufficiency (creatinine >2.0 mg/dL), untreated hypothyroidism, systemic lupus erythematosus, leukemia, or psoriasis. They also were excluded if they had any changes in doses of drugs known to affect Hcy levels during the 3 months preceding the study (methotrexate, phenytoin, carbamazepine, theophylline, nicotinic acid, or fenofibrate). Potential subjects were not allowed to take B-vitamin or FA-containing preparations other than those prescribed in the study protocol. Individuals using FA supplements or multivitamins (MVs) containing FA stopped taking them at least 10 weeks before screening.

Coronary heart disease was defined as a history of myocardial infarction, coronary artery revascularization, or current angina. Hypertension was defined as a systolic blood pressure ≥140 mm Hg or current use of antihypertensive medication. Diabetes mellitus was defined as a history of diabetes mellitus or use of an antiglycemic medication.

Study design 

After a 10-week placebo run-in (placebo phase I), all subjects were treated with a MV (Nature Made Mature Balance Multiple Vitamin and Mineral Supplement, Pharmavite Corp, Mission Hills, Calif) containing 400 μg FA, 6 mg vitamin B₆, and 25 μg vitamin B₁₂ daily for 10 weeks, followed by placebo washout for 10 weeks (placebo phase II) (Figure 1). Subjects then received both an MV and an FA (1 mg) supplement (total FA, 1400 μg) daily for 10 weeks (Figure 1). The 10-week treatment duration chosen for this study fell within the range used for similar studies evaluating the effects of FA and B vitamins on endothelial function in middle-aged adults (6 to 16 weeks)¹⁴, ¹⁵, ¹⁶, ¹⁷, ²⁴, ²⁵, ²⁶ and on plasma Hcy levels in older adults (6 to 12 weeks).¹⁰, ¹¹, ¹² Brachial artery reactivity, dietary folate intake, and laboratory values were measured at each visit (baseline, 10 weeks, 20 weeks, 30 weeks, and 40 weeks). Subjects fasted for at least 12 hours before each visit. Dietary folate intake was assessed through the use of a 15-item food frequency questionnaire, developed for this study, with possible scores ranging from 0 to 45 points (0 to 3 points for each food item assessed). Dietary folate intake scores from 0 to 15 points represented low intake, 16 to 30 points represented moderate intake, and 31 to 45 points represented high intake. Study drug compliance was assessed at each visit by pill counts. Laboratory tests performed at each visit included total plasma Hcy, serum vitamin B₁₂, vitamin B₆, folate, red blood cell (RBC) folate, and creatinine. All subjects had a thyroid-stimulating hormone level and lipid profile measured at the baseline visit. During this study, the MV manufacturer reduced the vitamin B₆ content from 6 mg to 4 mg (Nature Made Essential 50+ Multiple Vitamin and Mineral Supplement). The FA and vitamin B₁₂ contents did not change.

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• Figure 1. 

  Study design, flow, and medication changes.

Laboratory measurements 

Total plasma Hcy was measured by high-performance liquid chromatography (Beckman Coulter Inc, Fullerton, Calif), in which the plasma sample was treated with tris-carboxyethyl-phosphine (Sigma Chemicals Pty, Ltd, Perth, Australia) to ensure that all the Hcy was free and reduced.²⁷, ²⁸, ²⁹ Hcy was derivatized through the use of standard reagents (SBD-F, Fluka Chemicals, Buchs, Switzerland) and quantified by reverse phase high-performance liquid chromatography.²⁷, ²⁸, ²⁹ For normal Hcy levels (8.18 ± 0.19 μmol/L), the coefficient of variation was 2.40%. For elevated Hcy levels (20.52 ± 0.53 μmol/L), the coefficient of variation was 2.60%.

Serum vitamin B₁₂, serum folate, and RBC folate levels were measured by chemiluminescent enzymatic immunoassay. Serum vitamin B₆ levels were measured by radioenzymatic assay and serum creatinine by a kinetic alkaline picrate method. Serum lipid levels were measured by standard enzymatic techniques.

Brachial artery reactivity 

B-mode ultrasound was used to measure FMD of the brachial artery at each visit. Investigators and sonographers were blinded to subject treatment phase, age, and laboratory values when acquiring and measuring vessel diameters and calculating FMD. Brachial artery diameters and blood flows were imaged with a 7.5-MHz linear array vascular ultrasound transducer and a Sonos 5500 Ultrasound system (Philips Medical Systems, Andover, Mass). Increased forearm blood flow was induced by inflating a blood pressure tourniquet around the widest part of the forearm to a systolic blood pressure of 250 mm Hg (or 50 mm Hg greater than the systolic pressure) for 4.5 minutes. Repeat brachial artery diameter and blood flow scans were obtained immediately and 1 minute after tourniquet deflation. As a control, at the first and last visits (weeks 0 and 40), resting brachial artery diameter and blood flow scans were repeated after a 10-minute recovery period. Sublingual nitroglycerin (400 μg) was administered, and final scans were performed after 3 minutes.

Longitudinal images were recorded by using the digital storage and retrieval software of the ultrasound system.³⁰, ³¹ Mean vessel diameters were measured at end-diastole in triplicate by tracing a 1-cm segment (AccessPoint, Freeland Systems, LLC, Indianapolis, Ind), using media-adventitia interfaces (M-line) as landmarks. FMD was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter, expressed as a percent change. Nitroglycerin-mediated vasodilation (NTGMD) was calculated in an analogous fashion. Data from our laboratory showed that intraobserver reliability for measurement of the brachial artery diameter was 0.987, reflecting an interclass correlation coefficient across all readings and conditions.³⁰

Statistical analysis 

Clinical and brachial artery parameters were described by means and SEM, as well as medians and interquartile ranges. Outcome parameters included changes in FMD, NTGMD, and Hcy values. Changes after treatment with MV (week 20 vs week 10) were assessed by paired t tests, as were changes after MV + FA supplementation (week 40 vs week 30). Pearson correlation coefficients described linear relations between parameters. The determinants of change in FMD, NTGMD, and Hcy levels were assessed by univariate and multivariate linear regression, with baseline vessel diameter, creatinine, vitamin B₆, and B₁₂ levels and dietary folate scores as independent variables.

Sample size estimations were based on published sample-size nomograms and data from our laboratory.³⁰, ³¹, ³² Both treatment phases of this study had >80% statistical power to detect an absolute change of 2% in FMD and >90% power to detect a 2-μmol/L change in Hcy levels (α = 0.05).³⁰, ³¹, ³²

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Results 

Baseline subject characteristics 

Twenty-seven subjects enrolled in the study. Twenty subjects (age, 78.0 ± 1.2 years; range, 70 to 88 years; 9 women) completed the MV intervention and 17 subjects (age, 78.5 ± 1.3 years; 7 women) completed the MV + FA intervention. Figure 1 outlines reasons for attrition. Baseline characteristics are described in Table I. Before the 10-week washout period, 5 participants had used supplements. Medication changes that occurred during the study are shown in Figure 1. Dietary folate scores were in the low intake range at baseline and decreased slightly during MV administration (week 10 vs week 20: 12.7 ± 0.9 vs 10.6 ± 0.9 points, P = .018) but did not change significantly during the MV + FA period (week 30 vs week 40: 11.8 ± 0.9 vs 11.4 ± 0.9 points, P = .496). The study drug compliance rate was 97% ± 1%.

Table I. Baseline characteristics (week 0)

Values are number (%) or mean ± SE (median, IQR₂₅–IQR₇₅).

Laboratory results 

At baseline, Hcy levels were elevated (12.8 ± 0.6 μmol/L) and correlated positively with creatinine (r = 0.65, P = .003) (Table II). After 10 weeks of MV supplements, serum folate, vitamin B₆, and vitamin B₁₂ levels increased (P < .001). Hcy levels tended to decrease (12.8 ± 0.6 vs 12.0 ± 0.5 μmol/L, P = .056). After a 10-week washout period (placebo phase II), Hcy, vitamin B₁₂, and RBC folate levels did not change (all P > .080), whereas serum folate and vitamin B₆ levels declined (P < .001) (Table II). After 10 weeks of MV + FA supplements, serum folate, RBC folate, and vitamin B₆ levels increased (P < .001) (Table II); however, only a small change in Hcy levels was observed (11.7 ± 0.7 vs 11.4 ± 0.7 μmol/L, P = .778). Women and men had similar changes in Hcy levels with both interventions. During the 40-week study, Hcy levels decreased by 1.4 ± 0.9 μmol/L (p_trend = .034, Figure 2). Creatinine levels did not change.

Table II. Effects of multivitamins and multivitamins plus folic acid on laboratory and brachial artery ultrasound parameters

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• Figure 2. 

  Effects of MVs and MVs + FA on Hcy levels.

Brachial artery reactivity results 

At baseline, FMD (2.0% ± 1.2%) and NTGMD (10.2% ± 1.8%) were impaired compared with previously reported normal values for healthy middle-aged and older adults (Table I).¹ After 10 weeks of MV, mean FMD did not change significantly (3.0% ± 0.9% vs 2.4% ± 1.1%, P = .716) (Table II). After controlling for baseline brachial artery diameter, creatinine, B-vitamin levels, and dietary folate score, these changes remained statistically nonsignificant. After 10 weeks of MV and FA supplementation, FMD also did not change significantly (2.6% ± 0.9% vs 1.9% ± 0.7%, P = .484) (Table II). Neither FMD nor NTGMD changed at week 40 when compared with baseline (Figure 3). Women and men had similar FMD responses with both interventions.

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• Figure 3. 

  Effects of MVs and MVs + FA on FMD of the brachial artery.

Adverse events 

Two subjects died during this study (Figure 1). Another subject died from newly diagnosed hepatocellular carcinoma shortly after he withdrew from the study for personal reasons in placebo phase I. None were believed to be related to study participation.

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Discussion 

This study showed that 10 weeks of treatment with a MV or MV + FA supplements at doses commonly used in clinical practice did not improve FMD in adults ≥70 years old with mild hyperhomocysteinemia. Hcy levels decreased over the 40 weeks of this study, although the decreases observed during the low- and moderate-dose FA supplementation periods were not statistically significant. To our knowledge, this is the first study to evaluate specifically the effects of Hcy-lowering therapy on endothelium-dependent vasodilation in adults ≥70 years old.

At baseline, subjects in this study had impaired vascular reactivity, consistent with previous studies of older individuals with risk factors for CHD.³¹ Although significant advances in understanding vascular aging have been made over the last decade,³ little is known about the ability of medical therapies to reverse the age-related processes of endothelial dysfunction, arterial wall thickening, and impaired arterial compliance. Therapies targeting specific vascular risk factors, such as dyslipidemia, may improve endothelial function in middle-aged adults but may have little or no effect on vascular reactivity in geriatric patients.³¹, ³³, ³⁴, ³⁵ Increased vascular smooth muscle cell proliferation and impaired arterial compliance may limit the effectiveness of these therapies to improve FMD, since these processes are interrelated.³⁶ NTGMD, which reflects endothelium-independent vasodilation, also did not change after supplementation with FA and B-vitamins. Since many therapies used to prevent CHD have pleiotropic effects, treatments that may not affect vascular function in older adults still may have clinical benefits through effects on inflammation, thrombosis, and other mechanisms. For example, lipid-lowering therapy clearly prevents adverse cardiovascular events but does not improve FMD in older patients.³¹, ³⁷, ³⁸

Participants had mildly elevated Hcy levels, so the results may not be applicable to individuals with more severe hyperhomocysteinemia. Although the literature suggests that 30% of adults ≥67 years of age had hyperhomocysteinemia (Hcy >14 μmol/L) before 1998,⁴ after screening 93 adults for our study, we found that only 6% had Hcy levels >14 μmol/L.³⁹ Lower-than-expected Hcy levels may be related to United States Government–mandated FA supplementation of cereal grain flour, a finding confirmed by data from the Framingham Heart Study.⁴⁰ Thus, clinicians may see greater numbers of geriatric patients with mild rather than severe hyperhomocysteinemia, making our results more applicable to the general geriatric population. Although nearly all of our subjects had hypertension, nearly 75% of older adults have elevated blood pressure,⁴¹ so our study population reflects the makeup of patients seen in clinical settings.

This study was designed to evaluate the effects of doses of MV and FA supplements commonly used in clinical practice. Since optimal doses of FA and B-vitamin supplements are unknown, this study does not exclude the possibility that higher supplemental doses or a longer duration of treatment are needed to improve endothelial function in older persons.¹², ²², ²³ Several studies using higher doses of folic acid supplements or longer treatment duration, however, did not show improvement in FMD, including one that enrolled participants between the ages of 46 to 79 years (mean age, 63 years).²⁴, ²⁵ Furthermore, the effects of folic acid on endothelial function may be independent of its effects on plasma homocysteine levels.²⁶

Although some data support the potential beneficial effects of Hcy-lowering therapy on vascular reactivity and clinical cardiovascular outcomes, there is increasing evidence that challenges the clinical utility of FA and B-vitamin supplements. Indeed, preliminary data from two recent clinical trials suggest possible harm from long-term folic acid supplementation.⁴², ⁴³ The Cambridge Heart Antioxidant Study-2 (CHAOS-2) studied the effects of high-dose FA (5 mg daily) and placebo on nonfatal myocardial infarction, cardiovascular death, or unplanned revascularization in 1882 adults with ischemic heart disease. Despite a reduction in Hcy levels, preliminary data showed no reduction in cardiovascular events and a trend toward increased risk of nonfatal myocardial infarction with FA supplements.⁴² In a recent trial of 636 adults who had undergone coronary stenting, folate and B vitamin supplementation was associated with significantly smaller minimum lumen diameters after 6 months compared with the placebo group.⁴³ These findings partially contradict the Swiss Heart Study, in which subjects taking FA and B-vitamin supplements had lower restenosis rates.⁴⁴, ⁴⁵ Of interest, when the subjects in the Swiss Heart Study were divided into tertiles based on their total and LDL-C levels, only those in the highest tertiles had a reduction in the need for target lesion revascularization after folate supplementation.⁴⁵ These data support our findings suggesting a lack of effect of Hcy-lowering therapy in individuals with lower total and LDL-C levels. These studies and our findings underscore the importance of clinical trials testing the effects of Hcy-lowering on cardiovascular outcomes across the spectrum of ages.

Limitations 

Although the sample size in this study was small, each subject served as their own control. The data used for power estimates included published nomograms as well as data from our own laboratory.³⁰, ³¹, ³² This study did not screen for the C677T mutation in the methylenetetrahydrofolate reductase gene, which is associated with increased Hcy levels and greater reductions after FA supplementation; however, this genetic mutation does not appear to affect the responsiveness of endothelial function to folate supplementation.²⁵, ⁴⁶, ⁴⁷

During the MV intervention, dietary folate scores decreased slightly, so decreased dietary folate intake could have affected the total daily intake of FA by participants; however, subjects had low dietary folate intake (scores <15 points) at baseline and throughout the study. Furthermore, despite this slight decrease in dietary folate intake scores, serum folate levels increased during the MV treatment phase. Dietary folate scores were low and did not change during the MV + FA intervention phase. Although the manufacturer reduced the amount of vitamin B₆ in the MV (from 6 mg to 4 mg), it is unlikely that this small change affected the study results. Indeed, the changes in vitamin B₆ levels after MV and MV + FA supplementation were not different between the subjects assigned to the two different vitamin preparations. Furthermore, changes in FMD and laboratory parameters after MV and MV + FA supplementation did not differ significantly between the two MV preparation groups.

Other medications initiated during the study (Figure 1) may have affected study results; however, they only affected a few patients. Niacin may increase Hcy levels and improve FMD; however, the single subject who began niacin had a subsequent reduction in his Hcy level and worsening of his FMD during the intervention phase, suggesting that the niacin had little effect.⁴⁸, ⁴⁹ β-Blockers have not been shown to affect FMD.⁵⁰ One participant started taking atorvastatin, had his dose of atenolol increased, and began a long-acting nitrate; however, the net outcome of these medication changes on FMD was not significant.

Conclusions 

At doses commonly used in clinical practice, low- and moderate-dose FA and B-vitamin supplements do not improve brachial artery FMD in older adults with mild hyperhomocysteinemia. Clinical trials are needed to assess the effects of higher doses of supplements and longer treatment duration. To evaluate the effects of Hcy-lowering therapy on clinically important outcomes, randomized, controlled trials with clinical cardiovascular end points conducted in older adults will be required.

We thank all of the dedicated study participants.

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