Q J Med 2002; 95: 27-35
© 2002 Association of Physicians
The effect of folic acid supplementation on plasma homocysteine in an elderly population
From the Departments of General Practice and Primary Care and 1 Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK
Received 9 May 2001 and in revised form 17 October 2001
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Background: Increased plasma homocysteine is associated with coronary artery disease, peripheral vascular disease and venous thrombosis. Folic acid is the most effective therapy for reducing homocysteine levels. The lowest effective supplement of folic acid is not known, particularly for the elderly who have the highest prevalence of these conditions.
Aim: To explore the effects of daily supplements of 0, 50, 100, 200, 400 and 600 µg folic acid on plasma homocysteine in an elderly population.
Design: Randomized double-blind placebo-controlled trial.
Methods: Participants (n=368) aged 65–75 years were randomly allocated to receive one of the treatments for 6 weeks. Plasma homocysteine was recorded after 3 weeks and 6 weeks of supplementation.
Results: Only the 400 µg and 600 µg groups had significantly lower homocysteine levels compared to placebo (p=0.038 and p<0.001, respectively). Using multiple linear regression and each individual's total folic acid intake (diet plus supplement), a total daily folic acid intake of 926 µg per day would be required to ensure that 95% of the elderly population would be without cardiovascular risk from folate deficiency.
Discussion: A daily folic acid intake of 926 µg is unlikely to be achieved by diet alone. Individual supplementation or fortification of food with folic acid will be required to reach this target.
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Introduction |
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Elevated homocysteine is an independent risk factor for coronary artery disease, peripheral vascular disease and thrombosis.1–5 Elevated homocysteine concentrations have been found in 40% of patients with vascular disease2 and 35% of patients with venous thromboembolism.6 The Physicians Health Survey7 demonstrated prospectively that raised homocysteine antedates the vascular disease and is not simply a consequence of disease, although in this selected population of American physicians, the proportion of subjects with antecedent raised plasma homocysteine was much lower (13%) than in case-control studies of established disease. In a recent publication from the Framingham study, 1933 elderly participants (mean age 70±7 years) were followed up for a median of 10 years. Proportional hazards modelling revealed that homocysteine
14.26 µmol/l (the upper quartile) vs. <14.26 µmol/l (lower three quartiles) was associated with relative risk estimates of 2.18 (95%CI 1.86–2.56) for all causes and 2.17 (95%CI 1.68–2.82) for cardiovascular disease mortality. After adjustment for age, sex, systolic blood pressure, diabetes, smoking and total and high-density lipoprotein cholesterol, the relative risk estimates remained significant, 1.54 (95%CI 1.31–1.82) for all-cause mortality and 1.52 (95%CI 1.16–1.98) for cardiovascular mortality. It was concluded that elevated non-fasting homocysteine levels are independently associated with increased mortality rates in the elderly.8 Diet-induced hyperhomocysteinaemia may be the cause of this increased vascular risk, probably acting synergistically with conventional risk factors.5 In the Framingham cohort, there is a clear relationship between homocysteine level and both folate intake and plasma folate concentration.9 Nutritional studies have shown an inverse correlation between homocysteine and plasma concentrations both of vitamin B12 and folate.10
In the USA, 40% of an elderly population was folate-deficient, and of subjects with a low folate level, 84% had a raised homocysteine.9 The prevalence of high homocysteine levels in the elderly in Belgium, the Netherlands and Germany was found to be between 30% and 50%, although only 5–19% had abnormal folate or B12 levels. No studies have looked at the impact of folic acid supplementation in this age group, who probably have higher requirements and a lower dietary intake than a younger population. There is, therefore a particular need for definition of the recommended daily intake of folic acid and level of food fortification required by the elderly.
Folic acid is the single most effective therapy for hyperhomocysteinaemia.11 The effective daily dose of folic acid for achieving normal homocysteine levels in younger subjects is about 400 µg.1,12,13 Higher doses are no more effective.11 Folic acid reduces homocysteine level at all pre-supplementation levels; however, the reduction achieved is influenced by the pre-treatment level, so that those with higher initial homocysteine levels have the largest decrease (39% where homocysteine >18.5 µmol/l vs. 16% where pre-treatment homocysteine <8.9 µmol/l).11 The effectiveness of doses <400 µg has not been explored. This is important for public health policy; a modest dietary folic acid increase could be achieved by dietary modification, whereas higher intakes could only be achieved by food fortification or individual supplementation. Most studies have been carried out in young adults. The effect of folic acid supplementation has not been confirmed in the elderly.
The aim of this study was to determine whether manipulation of folate intake within the normal range reduces plasma homocysteine to levels thought to be free of cardiovascular risk in a healthy elderly population.
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Methods |
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Study design
This was a randomized, double-blind, placebo-controlled trial with a placebo run-in phase.
Subjects
Eligible participants were drawn from the registered population of two practices in Aberdeen. They were aged between 65 and 75 years when the lists were generated in January 1997. Research nurses screened the medical records and excluded individuals who were taking drugs known to interfere with folate, vitamin B6 or vitamin B12 metabolism (anti-epileptic, anti-metabolite, triamterene, trimethoprim, hydralazine, cimetidine); with major liver or renal disease; taking vitamin supplements containing folic acid; with megaloblastic anaemia; who had had previous gastric surgery; who had major psychiatric illness, who had unstable medical conditions, or who were unable to give informed consent. Patients who were thought to be unable to attend the clinic due to mobility problems were also excluded due to practical reasons for the running of the trial. All eligible individuals were sent a letter from their general practitioner informing them about the study followed by a telephone call from one of the research nurses to invite them to attend for the first visit. Those found to have abnormal screening blood tests at the first visit, especially a low vitamin B12 level, were also excluded at this stage. The local Joint Ethical Committee approved the study and all participants provided their written informed consent.
Tablets, identical in appearance, containing 0 (placebo), 50, 100, 200, 400 and 600 µg folic acid were manufactured by Surepharm Pharmaceuticals. All tablets contained 47.5 mg sodium ascorbate to emulate a vegetable folic acid source and prevent oxidation. Tartrazine was added to the final blend to ensure that there was uniformity of colour. Preliminary analyses and stability testing at 30 °C and ambient room temperature was carried out to confirm folic acid content at the point of manufacture and for the duration of the treatment period.
Subjects were randomly allocated on an individual basis to receive one of the five doses of folic acid or placebo. A randomization schedule was generated using random number tables. At randomization, each subject was allocated the next available number. All investigators involved in the study remained blinded to the supplementation given to the study subjects until data analysis was complete.
At visit 1 (day 0), each subject's reported details, medical and drug history were recorded; these were subsequently checked with medical records. Subjects also had a clinical examination including blood pressure measurement, ECG, height, weight, skin fold thickness and arm span measurement. Blood samples were taken for full blood count, urea, creatinine, electrolytes, cholesterol, liver function tests, vitamin B12, folate, methylmalonic acid (MMA) and homocysteine levels. Elevated MMA is a sensitive marker of B12 deficiency.14 Subjects also completed the Scottish Heart Health/ MONICA questionnaire,15,16 including the food frequency section. Subjects were then given placebo tablets, instructed to take one tablet per day and asked to return in 21 days for visit 2.
At visit 2 (day 21), those subjects not excluded by blood test results (abnormal liver or kidney function or low vitamin B12 levels), completed the food frequency questionnaire section again and had further blood taken for homocysteine. Subjects were then randomized (see earlier) and supplied with their study medication from day 21 to 63. They were asked to return for visits 3 (day 42) and 4 (day 63). Further blood samples were taken for homocysteine at days 42 and 63 and for MMA on day 42. All visits were in the morning and all subjects were asked to attend after a light breakfast consisting of a cup of tea or coffee and a piece of toast spread with a small amount of butter or margarine and a teaspoon of jam or marmalade. This was to prevent artificially elevated homocysteine levels caused by a high protein breakfast. Dietary intake was assessed using the food frequency section of the Scottish Heart Health/MONICA questionnaire on days 0, 21 and 63. The dietary questionnaire was analysed using the Scottish Heart Health computer package.
Homocysteine samples were drawn in EDTA tubes and immediately stored on ice. They were centrifuged within 4 h of sampling. Samples of the supernatant were removed and stored at -20 °C until analysis. Total homocysteine was measured by high performance liquid chromatography with fluorescent detection using the method of Araki17 as modified by Ubbink et al.18 and Vester and Rasmussen.19 Standards were prepared in a low-homocysteine plasma pool by standards addition.
MMA was isolated from serum using gas chromatography according to the method developed by Rasmussen20 and derivatized using mass spectrometry according to Marcell et al.21
Calculation of sample size
Normal plasma homocysteine levels vary widely within populations. In the Framingham series of older subjects it was 11±3 µmol/l.9 Values >14 µmol/l are generally considered to be associated with atherosclerosis.7 To show a mean fall in plasma homocysteine of 2 µmol/l, which we consider clinically significant (based on the increased relative risk from epidemiological studies9,22), with a power of 90%, at 5% significance level, requires a minimum sample size of 48 subjects per group finishing the trial. To allow for a withdrawal/non-compliance rate of up to 20%, a sample size of 60 per group was selected.
Statistical analysis
The demographic variables of the subjects were compared between the groups using the 
2 test for categorical variables and one-way analysis of variance for continuous variables. Homocysteine levels were compared between the groups using repeated measures analysis of variance. Each of the different folic acid supplementation groups was compared to the placebo group. The variables ‘baseline MMA’ and ‘folic acid intake’ were chosen a priori to be included in the repeated measures analysis of variance as covariates.
Multiple linear regression with adjustment for the covariate MMA was performed (dependent variable, final homocysteine level; independent variable, total folic acid intake) to estimate the amount of folic acid required in the diet that corresponds to a homocysteine level of 10 µmol/l. To predict the total intake of folic acid required for 95% of individuals to have a homocysteine level of 10 µmol/l or less, 200 datasets were simulated using the parameter estimates found in the above regression, and the distribution of the residual error examined. For each dataset, the folic acid intake corresponding to a homocysteine level of 10 µmol/l was selected.
A p value <0.05 was considered statistically significant. Statistical analyses were done using SPSS for Windows, Version 8.
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Results |
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Thirty-five percent of the subjects considered for inclusion in the study were excluded after screening of their notes (see Table 1
). After randomization, three subjects withdrew from the study, one because of family problems, one who decided not to continue and one who was found to have a low B12 level after randomization (see Figure 1).
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The subjects randomized to different dose groups and those not eligible for the study were similar demographically and clinically (see Tables 2
and 3).
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Following log transformation, the homocysteine levels, MMA levels and folic acid intake were normally distributed. The distributions of these variables are expressed using geometric means and 95%CIs.
Homocysteine levels
Homocysteine levels were recorded on four occasions. Homocysteine-1 was the level measured during the screening visit; homocysteine-2 was measured after 3 weeks on placebo; homocysteine-3 was measured after 3 weeks of daily folic acid supplementation; and homocysteine-4 following 6 weeks of supplementation. The homocysteine-1 and homocysteine-2 levels are baseline values. The two baseline values did not differ statistically within the subjects (mean ratio of homocysteine-1/homocysteine-2=1.00, 95%CI 0.98–1.03, p=0.720). We therefore used the mean value of homocysteine-1 and homocysteine-2 as the baseline value for further analyses. Four of the subjects had homocysteine-1 values of <2; these samples were haemolysed. For these subjects only, the homocysteine-2 value was used to represent the baseline value. Prior to breaking the randomization code, three subjects were excluded from the dataset. One of these subjects had a homocysteine-3 of <2 with haemolysis; the second and third subjects had very high homocysteine-1 values and were considered statistical outliers. The distribution of the homocysteine levels for the six groups at baseline, and after 3 weeks and 6 weeks of supplementation are given in Table 4.
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Folic acid intake
Dietary folic acid intake was assessed twice during baseline and again after 6 weeks of supplementation. The computed folic acid intake was significantly higher at the first visit compared to the other two visits (see Table 5). As the intake did not differ between the second baseline reading and after 6 weeks of supplementation, we concluded that dietary folic acid intake had not changed during the course of supplementation. The folic acid intake recorded at the second visit was chosen to represent the subject's folic acid intake levels. The distribution of folic acid intake did not differ statistically between the groups (p=0.612, see Table 5). Folic acid intake was negatively correlated with baseline homocysteine levels (r=-0.159, p=0.002).
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MMA levels
Blood was taken for MMA levels at the first visit (screening) and the third visit (3 weeks of supplementation). For 13 of the study subjects, the MMA could not be analysed for technical reasons at one of these two visits. The MMA levels recorded before any supplementation was given (visit 1) did not differ between the six groups (p=0.644). The baseline MMA correlated positively with baseline homocysteine level (r=0.31, p<0.001). The MMA within an individual was significantly lower at the first visit than the third visit (mean ratio of MMA1/MMA3 (95%CI)=0.96 (0.93–0.996), p=0.027). However, this difference was entirely due to an increase in MMA in the group receiving the 400 µg supplement, and was not considered clinically significant (geometric mean -28.3 µmol/l (visit 1) vs. 32.1 µmol/l (visit 3), p=0.001, see Table 6).
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Comparison of supplementation groups to placebo
Baseline MMA and dietary folic acid intake were significantly associated with the baseline homocysteine, and therefore were adjusted for in the repeated measures analysis of variance. Additionally, a new variable was created representing the relative change in MMA between visit 1 and visit 3, and was included as a covariate in the analysis.
The repeated measures analysis of variance showed that the change in homocysteine of subjects receiving a supplement of 50 or 100 µg/day did not differ significantly from subjects receiving placebo (p=0.494 and 0.703, respectively). Subjects receiving 200 µg/day did show a trend over the 6 weeks for a reducing homocysteine level; however, this was borderline non-significant compared to the placebo group (p=0.075). Subjects who received supplements of 400 and 600 µg/day had significantly lower homocysteine levels compared to the placebo group (p=0.038 and p<0.001, respectively, Figure 2). When comparing the estimated marginal mean homocysteine levels of the different supplementation groups, those patients receiving placebo had homocysteine levels that were 1.23-fold higher (95%CI 1.11–1.38), 1.12 (1.07–1.25), 1.11 (0.98–1.23), 1.02 (0.90–1.14) and 1.04 (0.93–1.16) than the individuals receiving supplements of 600, 400, 200, 100 and 50 µg, respectively. Figure 3 shows the percentage of subjects who had homocysteine levels <10 µmol/l after 6 weeks of supplementation against the supplement dose.
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, Placebo; X, 50 µg; 
, 100 µg; 
, 200 µg; 
, 400 µg; •, 600 µg.
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Homocysteine levels versus total folic acid intake
For each individual, the total daily folic acid taken during the 6 weeks of supplementation (dietary intake at visit 2 plus supplement) was calculated, and multiple linear regression, with adjustment for baseline MMA, was performed. The average folic acid intake required to reduce the homocysteine level to <10 µmol/l in this population was 330 µg/day (Figure 4). As there was a relationship between baseline MMA and homocysteine, the individuals in this study may not be representative of individuals whose functional B12 status is normal. Therefore all homocysteine levels were adjusted using the relationship between MMA and homocysteine at baseline to the level each participant would have had if their B12 status was normal, i.e. to an MMA of 26.5 µmol/l. This equated, by regression, to a homocysteine of 10 µmol/l (Table 4
). When the multiple regression is re-run using the MMA adjusted homocysteine level, the average total folic acid intake required to achieve a homocysteine of 10 µmol/l is 289 µg/day. This intake represents the average folate requirement to achieve a homocysteine of 10 µmol/l or less if the population B12 status is normal.
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However, there was a wide variance in the relationship between folate intake and plasma homocysteine level, and such an intake would mean that half the population had homocysteine levels of >10 µmol/l. The total folic acid intakes required to ensure that 75%, 95% and 97.5% of the population have homocysteine levels below 10 µmol/l, if the vitamin B12 status is as it is within our population, and with a normal vitamin B12 status, are presented in Table 7
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Discussion |
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We deliberately studied the elderly (65–75 years) section of the population because they have a higher dietary requirement than younger people and also carry the major burden of cardiovascular disease. Bots et al.23 studied subjects aged over 55 years old and found that in subjects aged 55–74 years, elevated homocysteine levels are associated with an increased risk of atherosclerosis and cardiovascular disease. They found no such association in those aged >75 years. Any public health policy designed to ensure adequate folate intake should therefore specifically address this age group. Data derived from younger subjects may not adequately address the elderly, whereas a policy designed for the elderly is likely to more than cover the requirements of younger age groups.
Following 6 weeks supplementation, only participants receiving the highest dose of 600 µg had unequivocally lower homocysteine levels compared to the placebo group (p<0.001, see Figure 2). The fall in homocysteine was greater in the first 3 weeks of supplementation for the subjects receiving 400 and 600 µg. Six weeks of supplementation may not have been long enough to reach a steady state in this population, and the 200 and 400 µg groups may have had a further reduction if observed for longer. Both these levels brought about a reduction at around the level of significance. In younger subjects, a dose of 400 µg folic acid is effective at lowering homocysteine levels.1,24
There is currently no firm basis for recommending a specific cut-off point for homocysteine that minimizes the cardiovascular risk. The risk associated with homocysteine is continuous across the concentration distribution.1,3,5,25 Graham et al.5 found that risk began to rise from the middle of the distribution (10.3 µmol/l). Malinow et al.25 found the referent level (OR=1) was 9.8. The referent value for risk of death associated with homocysteine was >9.0 µmol/l26 or >10.0 µmol/l.27 Thus, we chose a therapeutic goal of basal homocysteine <10 µmol/l for our analyses, rather than the definition of normal based on population statistical values of mean±2SD. Choosing as a goal for 95% of the elderly population to have a homocysteine of 10 µmol/l or less, would require a total folic acid intake of 1054 µg/day if the vitamin B12 status was as in our subject group, or 926 µg/day for a theoretical population with no B12-deficient individuals. Such high intakes of folic acid are unachievable through diet alone. Our analysis indicates that individual supplementation or food fortification with folic acid will be required to reach this intake and remove folate-responsive hyperhomocystinaemia as a cardiovascular risk factor from 95% of the population.
The above recommended level of folic acid intake assumes that the dietary intake has been accurately assessed. The mean folic acid intake for 65–74 year olds in the National Diet and Nutrition Survey 199828 was 282 µg/day for men and 215 µg/day for women. In our study, we observed at baseline similar levels for men (median 305 µg/day) but higher levels for women (median 308 µg/day). The dietary questionnaire used in this study assumed standard portion sizes derived from a younger population. This is because there are no data on portion size in this age group. Therefore our daily recommendation of folic acid intake could be over-estimated in the female population. Dietary folic acid intake was measured three times. The computed folic acid intake was significantly higher at the first visit (mean 15 µg/day) compared to the other two visits. In the absence of any logical explanation for this we elected to choose the second visit to represent the subjects' daily dietary folic acid intake as opposed to the higher values recorded at visit 1. Therefore our daily recommendation for total folic acid intake could be underestimated by a mean of 15 µg/day.
Thirty-five percent of patients on the practice lists were excluded from the study. The majority of these patients were excluded from the study for logistical reasons, but they would be suitable to receive supplementation. However, it is unlikely there is any systematic difference between this group and the population studied in terms of folic acid intake or metabolism.
We specifically excluded all participants with a low plasma vitamin B12 from the study, on the grounds that these subjects would have high homocysteine levels that would not be expected to be responsive to, or benefit from, folate supplementation. A separate study is required to investigate such subjects.
Despite the fact that we excluded all subjects with an abnormal vitamin B12 level before the study, baseline MMA levels correlated positively with baseline homocysteine levels (r=0.3, p<0.001). This indicates that there should also be supplementation or fortification with vitamin B12 for the elderly to address borderline vitamin B12 deficiency that is not identified by measurement of plasma vitamin B12 level. Such an increase in intake may further reduce homocysteine levels and protect against masking borderline B12 deficiency by folic acid supplementation. Our data also raise the question of the sensitivity of blood vitamin B12 in the assessment of the functional vitamin B12 status and the need for MMA estimation to be more generally available for routine testing.
There is considerable epidemiological evidence for a relationship between plasma homocysteine and cardiovascular disease. Evidence that vitamin supplementation favourably affects the evolution of atherosclerosis is limited. To date, in one study29 of adults (n=38, mean±SD age 58±12 years) with mild hyperhomocysteinaemia, treatment with folic acid (2.5 and 5 mg/day) pyridoxine and vitamin B12 appeared to reduce the rate of progression of ultrasound-determined extracranial carotid artery plaque area after a mean follow-up of 4.4±1.5 years. Despite the potential for reducing homocysteine levels with diet and/or vitamin supplementation, further randomized placebo-controlled trials are required to investigate whether such a reduction reduces risk.
Our data suggest that, if the vitamin B12 intake is adequate, an intake of about 900 µg folate per day would be required to ensure that 95% of the elderly population would be without cardiovascular risk from folate deficiency. This corresponds to a daily supplement of 600 µg of folic acid. This is considerably higher than the level of fortification in the US, where 140 µg folic acid per 100 g cereal is estimated to increase the folic acid intake of most women by 80 µg per day. It is also higher than the level of fortification recommended in the COMA report of 240 µg/100 g of flour, which would increase average intake of folic acid by 201 µg/day.30 Whether the daily supplementation of folic acid is administered as a tablet or by food fortification is a policy issue for debate elsewhere.
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Acknowledgments |
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This study was supported by The Wellcome Trust project grant (045122/Z/95/Z/MP/JF). We thank C. Harris, B. O'Hanrahan and K. Jack for assistance with patient recruitment and assessments, R.S. Anderson and P. Mackie for assays, N. Clark for assistance with data entry, and the general practitioners and staff of Westburn and Elmbank Medical Groups.
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Notes |
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Address correspondence to Dr A. Rydlewicz, Department of General Practice and Primary Care, University of Aberdeen, Foresterhill Health Centre, Westburn Road, Aberdeen AB25 2AY. e-mail: a.rydlewicz{at}abdn.ac.uk
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