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Folate and vitamin B-12 and risk of fatal cardiovascular disease: cohort study from Busselton, Western Australia

Joseph Hung, associate professor ^a, John P Beilby, senior biochemist ^b, Matthew W Knuiman, associate professor ^c, Mark Divitini, research associate ^c. 

^a Departments of Cardiology and Medicine, Sir Charles Gairdner Hospital, University of Western Australia, Nedlands WA 6009, Australia, ^b PathCentre, QEII Medical Centre, Nedlands 6009, Australia, ^c Department of Public Health, University of Western Australia

Correspondence to: J Hung jhung{at}cyllene.uwa.edu.au

	Abstract

Top Abstract Introduction Participants and methods Results Discussion References

Objective: To test the hypothesis that the incidence of fatal coronary heart disease and cardiovascular disease in a general population is related to serum and red cell folate and vitamin B-12 concentrations.
Design: Cohort study with follow up of 29 years.
Setting: Busselton, Western Australia.
Participants: 1419 men and 1531 women aged 20 to 90 years, who were alive more than three years after their participation in the 1969 Busselton health survey. 2314 (78.4%) had no cardiovascular disease at the initial survey.
Main outcome measures: Hazard ratios for fatal coronary heart disease and cardiovascular disease in men and women according to baseline concentrations of serum and red cell folate and serum vitamin B-12.
Results: 213 men and 159 women died from coronary heart disease, and 342 men and 302 women died from cardiovascular disease. Serum and red cell folate concentrations showed a moderate positive correlation (r=0.26, P<0.001) but otherwise serum and red cell folate and serum B-12 concentrations were not strongly correlated with each other or with other standard risk factors. After age and standard risk factors were adjusted for, there was no independent association between folate and B-12 concentrations and death from coronary heart disease or cardiovascular disease in the full cohort or the subcohort with no cardiovascular disease at baseline. The multivariate adjusted hazard ratio for death from cardiovascular disease in the lowest versus the highest category of red cell folate concentration was 1.05 (95% confidence interval 0.77 to 1.43) in men and 1.10 (0.81 to 1.51) in women.
Conclusions: These findings do not support the hypothesis that lower folate and B-12 concentrations increase the risk of fatal cardiovascular disease in a general population. The routine use of these vitamins for preventing cardiovascular disease should await evidence from clinical trials.

What is already known on this topic Moderate hyperhomocysteinaemia is thought to be an independent risk factor for cardiovascular disease High homocysteine concentrations in the general population are mainly due to insufficient folate and B vitamin concentrations Evidence linking serum or dietary folate and B vitamin levels to incident cardiovascular disease is inconclusive What this study adds A large community cohort followed for 29 years showed no independent association of baseline serum and red cell folate and serum B-12 concentrations with mortality from cardiovascular disease Vitamin therapy to lower homocysteine concentrations should not be routinely recommended in the general population until the benefit is proved by controlled clinical trials

	Introduction

Top Abstract Introduction Participants and methods Results Discussion References

Epidemiological studies have shown that moderate hyperhomocysteinaemia is an independent risk factor for coronary, cerebral, and peripheral vascular disease. ^{1 2} Studies have also shown that moderately raised concentrations of homocysteine are prevalent in the general population and that an inverse relation exists between homocysteine concentration and concentrations of folate and vitamins B-6 and B-12. ^{3 4} Randomised clinical trials have shown that low dose vitamin supplementation, particularly with folic acid, significantly lowers homocysteine concentrations.⁵ However, there are no clinical trial data that prove that lowering homocysteine concentrations prevents deaths from cardiovascular disease or coronary heart disease.

Prospective evidence linking circulating or dietary levels of folate and vitamins B-6 and B-12 in the general population to incident cardiovascular disease remains limited and inconsistent.^6-11 The purpose of this cohort study was to test the hypothesis that an increased incidence of fatal coronary heart disease and cardiovascular disease was related to lower concentrations of serum and red cell folate and serum vitamin B-12 in a community population.

	Participants and methods

Top Abstract Introduction Participants and methods Results Discussion References

This study is based on 1772 men and 1904 women aged 20 to 90 years who attended the 1969 Busselton health survey, representing a 90% participation rate for the total adult population; cross sectional health surveys of adults listed on the electoral roll were undertaken in Bussleton, Western Australia, every three years from 1966 to 1981.

The methods of the surveys have been described elsewhere. ^{12 13} The participants were asked to complete a comprehensive health and lifestyle questionnaire and to undergo various measurements and tests (see bmj.com).

Outcome
Data on deaths were available to 31 December 1998. Deaths among survey participants were identified by linkage to the death register. Survival was confirmed by linkage to the electoral roll and Telecom White Pages and through relatives. We ascertained vital status at 31 December 1998 for 98% of the cohort. The survival times for the remaining 2% were censored at the last time known to be alive. The underlying cause of death was determined from the death certificates.

Analysis of data
There were 1486 men and 1570 women available for analysis after we excluded pregnant women and those with missing values for primary risk factors or adjustment variables. Of these, 67 men and 39 women died within three years of the 1969 survey and have also been omitted, leaving a total of 1419 men and 1531 women. A total of 1113 men and 1201 women (78.4% of cohort) had no history of coronary heart disease, leg claudication, or stroke at initial survey and were considered free from cardiovascular disease. We analysed data from this subcohort separately.

The risk factors of primary interest were serum and red cell folate concentrations and serum B-12 concentration. We also included other baseline risk factors for cardiovascular disease in the multivariate analysis: age, systolic and diastolic blood pressure, body mass index, serum cholesterol concentration, white cell count, smoking, menopause (in women), treatment for diabetes, treatment for hypertension, alcohol intake, and history of coronary heart disease, stroke, or leg claudication. ^{14 15}

We used Cox proportional hazards regression analysis of survival to death or end of follow up to assess the influence of primary risk factors after adjustment for age only and also after adjustment for age and other risk factors. ^{14 15} As the age adjusted and multivariate adjusted results were similar, only the multivariate results are reported. Vitamin concentrations were examined as continuous variables and also in categories.

	Results

Top Abstract Introduction Participants and methods Results Discussion References

At baseline the average age was about 48 years and the risk factor characteristics were typical of population samples surveyed around that time. Serum folate and red cell folate concentrations showed a moderate positive correlation (r=0.26; P<0.001) but neither serum nor red cell folate and vitamin B-12 concentrations were correlated with each other or with other risk factors.

After 29 years' follow up, 665 men and 537 women had died (excluding subjects who died within three years of initial survey). Death was due to cardiovascular disease in 342 men and 302 women and to coronary heart disease in 213 men and 159 women. In the subcohort of participants without cardiovascular disease, 475 men and 362 women had died; deaths were due to cardiovascular disease in 226 men and 187 women and to coronary heart disease in 131 men and 98 women.

Tables 1 and 2 show the multivariate adjusted hazard ratios for folate and vitamin B-12 quarters in relation to deaths from all causes, cardiovascular disease, and coronary heart disease. There was no consistent or significant pattern of relative risk for serum and red cell folate and serum B-12 concentrations in men and women in the full cohort. Among women in the subcohort without cardiovascular disease, there was a generally inverse association between serum folate concentrations and risk of death, but the only significant relative risk was for all cause mortality, and the trend P value was not significant at the 5% level.

Among men in the subcohort without cardiovascular disease, the trend model suggested an unexpected positive association between serum folate concentration and risk of death from cardiovascular disease (P=0.05), but the categorical model showed no association. When a subgroup of 85 men (7% of the disease-free cohort) with serum folate concentrations >9 µg/l and about double the risk of death from cardiovascular disease were removed from the analysis, there was no longer evidence of a trend for death from cardiovascular disease (P=0.66). There was no evidence of any other significant relation.

	Discussion

Top Abstract Introduction Participants and methods Results Discussion References

We found no evidence of an independent association between folate or vitamin B-12 concentrations and death from cardiovascular or coronary heart disease. The trends in risk factors and mortality in the Busselton population are similar to those in other parts of Australia.¹³ In addition, adjusted estimates of relative risk of coronary heart disease and stroke in the Busselton population seem consistent with estimates in other populations of similar age and ethnicity.^14-16

The 1969 Busselton health survey measured serum and red cell folate and serum vitamin B-12 concentrations as a means of estimating the nutritional status of the population.¹⁷ Folate and B-12 nutrition seemed to be generally sufficient, with only 3.1% of the population having a reduced folate and 0.4% having a reduced B-12 concentration based on the normal reference intervals for red cell folate (115-600 µg/l) and serum B-12 (160-850 ng/l).¹⁷ However, the absolute vitamin concentrations were unimportant as analysis was based on estimating relative risks.

Folate
Red cell folate indicates tissue folate status and reflects folate turnover over the preceding two to three months.¹⁸ It is a more reliable indicator of long term folate intake than serum folate, which has a high intraindividual variability because it reflects intake only in the preceding few days.¹⁸

Studies examining the association between serum or dietary folate concentrations and coronary heart disease have produced inconsistent results. Morrison et al found a significant inverse association between serum folate concentration and 15 year mortality from coronary heart disease, especially in women.⁶ However, they found no association between dietary folate consumption and risk of fatal coronary heart disease. Other cohort studies with follow up periods ranging from 3.3 to 20 years have found no significant increase in risk of non-fatal or fatal coronary heart disease for participants in the lowest versus highest group of serum folate concentration.^7-9 The high intraindividual variability of serum folate measurements may have diluted any observed association with risk of coronary heart disease in these studies as well as in our study.

By contrast, the US Nurses' health study¹⁰ and the Kuopio ischaemic heart disease risk factor study¹¹ of Finnish men found a significant inverse association between dietary folate intake and coronary heart disease events over 14 and 10 years respectively. However, these results need to be interpreted with caution as a high folate intake from food and vitamin supplements may also be related to other dietary factors or unmeasured risk behaviours that are independently associated with coronary heart disease.

Vitamin B-12
We found no association between vitamin B-12 concentration and death from cardiovascular or coronary heart disease. This may not be surprising given that vitamin B-12 supplementation has a relatively small effect on homocysteine concentration ^{4 5} and B-12 deficiency is uncommon in the general population, being usually related to a problem of absorption rather than nutrition.

Implications
Our negative findings together with the inconsistent results of previous cohort studies leave many question unanswered about the effect of folate and vitamin B-12 on homocysteine concentrations and risk of cardiovascular disease in the general population. The epidemiological data for homocysteine as a risk factor for cardiovascular disease is strong, and clinical trials have shown that homocysteine concentrations can be lowered by safe and inexpensive doses of folic acid and vitamin B-12.

Our results do not argue against public health efforts to raise folate consumption in the general population by increased intake of fruits, vegetables, and fortified grains and cereals.¹⁹ However, use of vitamin supplements to lower homocysteine concentrations should not be routinely recommended in the general population for prevention of cardiovascular disease until their benefit is proved by controlled clinical trials.

	Acknowledgments

We thank the Busselton Population Medical Research Foundation for access to the Busselton health survey data and the Busselton community for their longstanding cooperation and support for the Busselton health study.

Contributors: See bmj.com

	Footnotes

Funding: None.

Competing interests: None declared.

This is an abridged version; the full version is on bmj.com

	References

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(Accepted 26 September 2002)