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1 From the Northern Ireland Centre for Diet and Health, University of Ulster, Coleraine, United Kingdom, and the Departments of Clinical Medicine and Biochemistry, Trinity College Dublin.
2 Supported by EU Project PL 963549 and Abbott Germany. 3 Address reprint requests to H McNulty, Northern Ireland Centre for Diet and Health, University of Ulster, Coleraine BT52 1SA, United Kingdom. E-mail: h.mcnulty{at}ulst.ac.uk.
ABSTRACT
Background: Current data suggest that physiologic doses of vitamin B-6 have no significant homocysteine-lowering effect. It is possible that an effect of vitamin B-6 was missed in previous trials because of a much greater effect of folic acid, vitamin B-12, or both.
Objective: The aim of this study was to investigate the effect of low-dose vitamin B-6 supplementation on fasting total homocysteine (tHcy) concentrations in healthy elderly persons who were made replete with folate and riboflavin.
Design: Twenty-two healthy elderly persons aged 63–80 y were supplemented with a low dose of vitamin B-6 (1.6 mg/d) for 12 wk in a randomized, double-blind, placebo-controlled trial after repletion with folic acid (400 µg/d for 6 wk) and riboflavin (1.6 mg/d for 18 wk); none of the subjects had a vitamin B-12 deficiency.
Results: Folic acid supplementation lowered fasting tHcy by 19.6% (P < 0.001). After folic acid supplementation, baseline tHcy concentrations ranged from 6.22 to 23.52 µmol/L and 10 subjects had suboptimal vitamin B-6 status (plasma pyridoxal-P < 20 nmol/L). Two-way analysis of variance showed that the significant improvement in vitamin B-6 status in response to vitamin B-6 supplementation (on the basis of both pyridoxal-P and the erythrocyte aspartate aminotransferase activation coefficient) was reflected in a significant reduction in plasma tHcy of 7.5%.
Conclusions: Low-dose vitamin B-6 effectively lowers fasting plasma tHcy in healthy subjects who are both folate and riboflavin replete. This suggests that any program aimed at the treatment or prevention of hyperhomocysteinemia should include vitamin B-6 supplementation.
Key Words: Fasting homocysteine • vitamin B-6 • folic acid • riboflavin • plasma pyridoxal-P • erythrocyte aspartate aminotransferase activation coefficient • EASTAC • elderly
INTRODUCTION
Even mild elevations in total homocysteine (tHcy) are associated with an increased risk of cerebrovascular, peripheral vascular, and cardiovascular diseases (1). Once formed, homocysteine is either remethylated to methionine, which requires vitamin B-12 (2), folate (2), and riboflavin (3) as a cofactor, cosubstrate, and prosthetic group, respectively, or undergoes a transsulfuration reaction to form cysteine. The transsulfuration pathway is catalyzed by cystathionine ß-synthase, which is dependent on pyridoxal-P (PLP) (4).
Although vitamin B-6 has been used for many years to successfully treat homocystinuria caused by cystathionine ß-synthase deficiency (5), a role for vitamin B-6 in the prevention or treatment of mild hyperhomocysteinemia is unclear. Results from 9 studies that investigated the effect of vitamin B-6 (when it was given as a treatment on its own) on fasting plasma tHcy concentrations are inconclusive. Six of the aforementioned studies found no change (6–11) in fasting tHcy and one found a significant increase (12). To our knowledge, only 2 studies in the literature found a significant reduction in fasting tHcy after vitamin B-6 supplementation (13, 14). The first of these studies, carried out in 10 young women who had clinical and biochemical vitamin B-6 deficiency, found a significant lowering of fasting tHcy of 19.7% after 15 d of treatment with 20 mg pyridoxine hydrochloride/d. The trial, however, was not blinded or placebo-controlled and had no washout period. The second study (14), a recent placebo-controlled trial, showed a significant lowering of fasting tHcy of 17% (P < 0.011) in response to extremely large doses (120 mg/d) of vitamin B-6 in healthy subjects (n = 9).
Thus, the literature to date suggests either that vitamin B-6 does not have a homocysteine-lowering effect (6–12) or that it has a lowering effect only when given in exceptionally high doses (14) or in exceptional circumstances, ie, in pyridoxine-responsive homocystinuria (5), in selective patients with severe vitamin B-6 deficiency (13), or after methionine loading (6, 15, 16). This evidence may lead to the conclusion that in apparently healthy persons, a suboptimal vitamin B-6 status does not represent a general public health problem with respect to its effect on tHcy. This is at odds with the literature, which suggests that suboptimal vitamin B-6 status might represent a general risk for vascular disease (17–20), and with other studies that showed a significant inverse relation between tHcy and vitamin B-6 status (17–19, 21–24) and an accumulation of homocysteine in vitamin B-6 depletion-repletion studies (25, 26).
All of the above supplementation studies were carried out in volunteers aged 18–60 y and used pharmacologic doses of vitamin B-6. To date, no vitamin B-6 intervention has been carried out in elderly persons, even though it is well accepted that vitamin B-6 status declines with age (27, 28) and plasma tHcy concentrations increase with age (22, 29). Therefore, the aim of this study was to investigate the effect of low-dose vitamin B-6 supplementation on fasting tHcy concentrations in healthy elderly persons after exclusion of those deficient in vitamin B-12 and after optimization of folate and riboflavin status.
SUBJECTS AND METHODS
Subject recruitment
Ethical approval was granted by the Research Ethical Committee of the University of Ulster and subjects gave written, informed consent. Subjects aged 60 y were recruited between January and April 1998 through senior citizen groups and local folds (ie, sheltered accommodation providing no medical support for healthy elderly persons who live independently within a housing complex and take care of themselves). All potential subjects were interviewed by using a short medical questionnaire about the subjects' general health and drug and supplement use. The exclusion criteria were as follows: B vitamin supplementation, gastrointestinal disease, hematologic disorders, use of drugs known to affect vitamin B-6 or riboflavin metabolism (eg, antacids containing magnesium or ioniazid), impaired cognitive function [score < 7 on Hodkinson 10-Point Mental State Questionnaire (30)], serum creatinine concentration 105 µmol/L, serum vitamin B-12 concentration <111 pmol/L (150 ng/L), and vascular, hepatic, or renal disease.
Study design
The vitamin B-6 intervention was a 12-wk, randomized, double-blind, placebo-controlled trial. Subjects were randomly assigned to 1 of 2 groups to receive either vitamin B-6 or a placebo from a laboratory technician who was not involved with the study in any other way. Before starting the vitamin B-6 intervention, all subjects received supplementation with riboflavin (1.6 mg/d) for 12 wk followed by a combination of folic acid (400 µg/d) and riboflavin (1.6 mg/d) for an additional 6 wk. Folic acid and riboflavin supplementation continued throughout the vitamin B-6 intervention. At the start of the vitamin B-6 intervention, a 20-mL blood sample was taken after the subjects had fasted overnight. Subjects were then supplied with either vitamin B-6 (1.6 mg) or a placebo tablet daily. To maximize compliance, subjects were visited in their homes every 3 wk, at which time they were supplied with additional supplements; any unused tablets from the previous 3 wk were returned and counted. Subjects were instructed to maintain their usual diets and refrain from commencing any form of vitamin supplementation during the intervention.
Blood sampling
All blood samples were collected in the subjects' homes while they were in a sitting position. Three tubes of blood were collected from each subject: one 8-mL EDTA-treated tube for plasma and red blood cell extraction, one 4-mL EDTA-treated tube for preparation of red blood cell lysate and measurement of packed cell volume, and one 8-mL serum separation tube for serum extraction. The tube containing plasma for homocysteine, vitamin B-6 (plasma PLP), erythrocyte aspartate aminotransferase activation coefficient (EASTAC), and riboflavin (erythrocyte glutathione reductase activation coefficient, EGRAC) measurements was wrapped in foil and placed on ice immediately after collection and centrifuged within 0.5–2.5 h at 719 x g (2000 rpm) for 15 min at room temperature to separate plasma and red blood cells. After centrifugation, the plasma layer was removed and stored. The remaining red blood cells were washed 3 times with phosphate-buffered saline. The saline and buffy layer were removed after each centrifugation and the resulting washed red blood cells were stored. A lysate of red blood cell folate was prepared from the 4-mL tube by diluting blood 1 to 10 with a freshly prepared 1% ascorbic acid solution. The packed cell volume (required for the calculation of red blood cell folate concentrations) was measured in the remaining whole blood by using an automated Coulter counter (Coulter Electronics, Hialeah, FL). Serum separation tubes were centrifuged at 719 x g (2000 rpm) for 20 min at room temperature and the serum layer was removed. All preparations were stored at -70°C for batch analysis at the end of the study.
Biochemical measurements
Plasma tHcy was measured by immunoassay (31), plasma PLP was determined by reversed-phase HPLC with fluorescence detection (32), and red blood cell folate (33), serum folate (33), and serum vitamin B-12 (34) concentrations were measured by microbiological assay. EASTAC (35) and EGRAC (36) were measured by enzyme assay with the Cobas Fara centrifugal analyzer (Roche Diagnostics, Welwyn Garden City, United Kingdom). For all assays, samples were analyzed blind, in duplicate (except for EGRAC, for which triplicate samples were measured), and within 6 mo after sampling. Quality control was provided by repeated analysis of stored batches of pooled erythrocytes (for EASTAC and EGRAC), plasma (for tHcy and plasma PLP), serum (for vitamin B-12 and folate), and lysates of red blood cell folate (for red blood cell folate) covering a wide range of values.
Dietary intake and anthropometric data
Current dietary intake was assessed with use of a 4-d estimated food record. Portion sizes were estimated by the subjects using household measures. Volunteers were required to keep the food records from Saturday to Tuesday to ensure that both weekdays and weekend days were included. All volunteers received comprehensive instructions on how to complete the food records from a trained nutritionist (MCM), who also subsequently quantified portion sizes using published food portion sizes (37) and calculated nutrient intakes using the nutrient analysis program COMP-EAT (Lifeline; Nutritional Services Ltd, Grantham, United Kingdom). Body mass index (BMI) was calculated as weight (in kg)/height2 (in m). The basal metabolic rate (BMR) was calculated by using the formulas of Schofield (38), which are based on sex, height, and weight for the age groups 60–74 y and >74 y. The ratio of BMR to energy intake (obtained from the 4-d estimated food record) was calculated on an individual basis to identify likely underreporters of food intake according to the statistically derived cutoff limits of Goldberg et al (39).
Statistics
All statistical analyses were performed by using the SPSS Statistical Package for the Social Sciences (version 9.0.1; SPSS UK Ltd, Chersey, United Kingdom). Correlation analysis was performed by using bivariate Pearson correlation coefficients. Baseline values for the placebo and vitamin B-6 groups were compared by using an independent-samples t test. Responses to intervention within and between groups were examined by using two-way (time and treatment) ANOVA; P values < 0.05 were considered significant.
RESULTS
Baseline characteristics and study compliance
On the basis of screening for vitamin B-12 deficiency at baseline, no individual was found to be vitamin B-12 deficient, ie, no subject had a serum vitamin B-12 concentration <111 pmol/L (150 ng/L). A total of 22 elderly volunteers with a mean age of 69.9 y participated in the vitamin B-6 intervention for 12 wk. There were no significant differences between the vitamin B-6 and placebo groups at baseline (Table 1). The compliance of all but one subject was excellent (one subject missed taking >2 tablets during the 12-wk supplementation period but was included in the analysis) and none of the subjects dropped out during the study. Baseline plasma tHcy concentrations ranged from 6.22 to 23.52 µmol/L; 20 of the 22 participants were classified as normohomocysteinemic at baseline (ie, plasma tHcy < 15 µmol/L; 40). Vitamin B-6 status was also assessed at baseline; 10 subjects were classified as deficient or suboptimal on the basis of a plasma PLP concentration <20 nmol/L and 6 were classified as deficient or suboptimal on the basis of an EASTAC value 1.80. There was no significant correlation between plasma PLP and EASTAC measurements. Serum vitamin B-12 (r = -0.437, P = 0.021) and red blood cell folate (r = -0.392, P = 0.035), but not serum folate, vitamin B-6 (on the basis of EASTAC or PLP), or riboflavin (on the basis of EGRAC) status, were significantly correlated with plasma tHcy at baseline (data not shown).
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TABLE 1. . General characteristics and biochemical status of subjects at baseline1
Baseline dietary data
Only one individual had a ratio of EI to BMR of <1.14; this individual was classified as an underreporter (39) and was not included in the dietary analysis. The mean dietary intake of the remaining group is shown in Table 2. The percentages of the population with dietary intakes of folate, vitamin B-6, vitamin B-12, and riboflavin below the reference nutrient intake (41) were 38%, 4.8%, 0%, and 19%, respectively. The dietary intake of all subjects, however, was above the lower reference nutrient intake. Plasma tHcy was not significantly correlated with the dietary intake of folate, vitamin B-6, vitamin B-12, or riboflavin (data not shown).
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TABLE 2. . Energy and B-vitamin intakes in free-living healthy elderly persons aged 63–80 y assessed with use of a 4-d food record
Intervention study
Folic acid supplementation, given before the vitamin B-6 intervention, significantly improved folate status, as indicated by a significant increase in serum folate (P < 0.001) from 15.27 ± 9.00 to 36.94 ± 12.71 nmol/L and a significant decrease in fasting plasma tHcy (P < 0.001) of 19.6%, from 12.65 ± 6.19 to 10.17 ± 3.86 µmol/L (data not shown). The responses of plasma tHcy and B-vitamin status to vitamin B-6 or placebo supplementation are shown in Table 3. Examination of individual responses to intervention showed one outlier in the placebo group (change in tHcy = -5.11 µmol/L compared with next largest change of 1.68 µmol/L). This outlier was excluded from the analysis. Two-way ANOVA showed that the significant change in vitamin B-6 status (on the basis of both PLP and EASTAC values) in the vitamin B-6 group compared with the placebo group was reflected in a significant lowering in tHcy of 7.5%. The change in serum or red blood cell folate status (as a result of continued supplementation with folic acid throughout the vitamin B-6 intervention) was not significantly different between the placebo and vitamin B-6 groups.
View this table:
TABLE 3. . Response of plasma total homocysteine (tHcy) and B-vitamin status to 12 wk of supplementation with 1.6 mg vitamin B-6/d in free-living healthy elderly persons1
DISCUSSION
The results of this study indicate that vitamin B-6 effectively lowers fasting tHcy concentrations in healthy elderly persons; however, the homocysteine-lowering effect is modest compared with that of folic acid [estimated to be 25% in a recent meta-analysis (42) and 19.6% in the present study]. The magnitude of the effect of folic acid compared with that of vitamin B-6 may differ because folate acts as a cosubstrate (2) whereas vitamin B-6 acts as a cofactor (4) in homocysteine metabolism. Alternatively, the magnitude of the effect may differ because the diet consumed currently by most people tends to result in a higher proportion of people with suboptimal folate rather than suboptimal vitamin B-6 status. Although both serum and red blood cell folate concentrations appeared to increase during the 12-wk vitamin B-6 supplementation period in both the placebo and vitamin B-6 groups (as a result of continued supplementation with folic acid), this increase in folate status did not affect plasma tHcy concentrations. Before intervention with vitamin B-6, plasma tHcy concentrations had reached a plateau as indicated by the lack of change in tHcy concentrations in the placebo group despite an apparent increase in red blood cell and serum folate concentrations.
The strengths of the present study were that it was double-blind and placebo-controlled and that the subjects were highly motivated and followed closely, which ensured excellent compliance. There are many possible reasons why vitamin B-6 intervention successfully lowered plasma tHcy in the present study but not in the other trials mentioned previously. First, all subjects in the present study were riboflavin replete, having taken riboflavin (1.6 mg/d) for a total of 18 wk before the vitamin B-6 intervention began. This may have been an important factor because the flavin mononucleotide–dependent conversion of vitamin B-6 to PLP by pyridoxamine-phosphate oxidase appears to be the limiting step in vitamin B-6 metabolism (43, 44). Therefore, in theory, vitamin B-6 supplementation in persons with suboptimal riboflavin status would be ineffective because adequate conversion of vitamin B-6 to the active form PLP would not occur and, thus, the transsulfuration pathway of homocysteine metabolism would not be stimulated. Second, all subjects in the present study were folate replete, having taken folic acid (400 µg/d) for 6 wk before the vitamin B-6 intervention began. This may have been an important factor in the activation of the transsulfuration pathway because high S-adenosylmethionine concentrations (as a result of enhanced remethylation resulting from folic acid supplementation) might activate the vitamin B-6–dependent transsulfuration pathway while inhibiting the remethylation pathway (45). A third possible explanation is that other studies may have missed a significant effect of vitamin B-6 because most of the elevation in homocysteine was due to suboptimal folate status. This being the case, the effect of vitamin B-6 supplementation may be significant only after all the homocysteine lowering in response to folic acid supplementation is accounted for. A final possible reason is that the present vitamin B-6 intervention was conducted in elderly subjects, whereas the studies that failed to show a homocysteine-lowering effect were not. Thus, we may have observed a significant homocysteine-lowering effect in response to vitamin B-6 even if we had not ensured the optimization of folate and riboflavin status in our subjects.
The effect of tHcy on vascular disease is graded (21, 23). Reductions in tHcy concentrations, therefore, are likely to be beneficial, even if tHcy concentrations are within reference ranges. If fortification with B vitamins is to be part of a public health strategy aimed at vascular disease prevention in a population, it is vital that the lowest effective dose and combination of B vitamins that will result in the greatest reductions in tHcy is known so that the risk of overexposure in those with the highest intakes of the fortified food is limited. Therefore, interventions undertaken to investigate the effect of very low doses of B vitamins on fasting tHcy concentrations, such as the present study, are vital.
Until the results of this study, vitamin B-6 appeared to lower homocysteine only in exceptional circumstances; therefore, many investigators involved in the design of secondary prevention trials (1) did not include vitamin B-6 supplementation in an effort to reduce the number of subjects required for these trials. Although the results of the present study need to be confirmed and investigated in other subgroups of the population, we suggest that it may be pertinent to include vitamin B-6 in any program aimed at the prevention or treatment of hyperhomocysteinemia. Additional work is also necessary to find the combination of B vitamins that most effectively lowers tHcy concentrations and to investigate the homocysteine-lowering capacity of riboflavin, which is intimately involved in homocysteine metabolism but has thus far received little attention. In conclusion, the results of the present study indicate that low-dose vitamin B-6 supplementation for 12 wk in highly compliant elderly subjects with optimal riboflavin and folate statuses effectively lowers fasting plasma tHcy concentrations.
ACKNOWLEDGMENTS
We acknowledge Clonmel Healthcare, Tipperary, Ireland, for providing us with the vitamin B-6 and folic acid supplements, and the volunteers, who kindly participated in this study.
REFERENCES