Evidence from observational studies

Clinical studies of patients with CVD published over the past 20 years provide much evidence to link low folate status and/or elevated plasma homocysteine with an increased risk of CVD. The data from numerous such studies, both retrospective and prospective, were used to generate two important meta-analyses, both published in 2002^(6, Reference Wald, Law and Morris⁷⁾. They predicted that lowering homocysteine by 3 µmol/l (or by about 25% based on typical homocysteine concentrations of 12 µmol/l) would reduce the risk of heart disease by 11–16% and stroke by 19–24%^(6, Reference Wald, Law and Morris⁷⁾. Apart from clinical studies, population data from North America showed an improvement in stroke mortality corresponding to the timing of the introduction of mandatory food fortification with folic acid in 1996–98 and no similar improvement over the same time period in the UK where no such fortification policy was put in place⁽Reference Yang, Botto and Erickson⁸⁾. Thus, the temporal decline in stroke-related mortality in the US and Canada which coincided with the introduction of folic acid fortification was consistent with a beneficial effect of folic acid or homocysteine-lowering on CVD⁽Reference Yang, Botto and Erickson⁸⁾. Randomised trials, however, were required in order to prove a cause-and-effect relationship.

Evidence from randomised controlled trials

Since 2004, the results of a number of much-awaited secondary prevention trials in at-risk patients began to appear in high-profile medical journals⁽Reference Toole, Malinow and Chambless^9–15). All these failed to demonstrate a benefit of homocysteine-lowering therapy on CVD events generally (Table 1). Some argued that these trials lacked sufficient statistical power to detect a beneficial effect, given that the magnitude of the association between homocysteine and CVD risk on re-evaluation of the epidemiological evidence was later found to be more modest than originally estimated at the time the trials were being designed⁽¹⁶⁾. The relationship for homocysteine and stroke, however, was always estimated to be stronger than that for heart disease^(6, Reference Wald, Law and Morris⁷⁾. In fact one of the aforementioned trials, the HOPE 2 trial⁽Reference Lonn, Yusuf and Arnold¹⁰⁾, had shown a clear benefit of B-vitamin intervention in reducing the risk of stroke, a result that for some reason was largely overlooked in the original report. Instead, the positive results of the trial in relation to stroke risk were separately reported 3 years after the original trial findings⁽Reference Saposnik, Ray and Sheridan¹⁷⁾, but these later results were not as widely recognised as the original report of the HOPE 2 trial. Of greater note, a meta-analysis of randomised controlled trials (RCT) showed that homocysteine-lowering by folic acid reduced the risk of stroke in general by 18%, but with significantly greater reductions observed (by 23–29%) in those trials of longer treatment duration or with greater homocysteine-lowering or in patients with no history of stroke (Table 2)⁽Reference Wang, Qin and Demirtas¹⁸⁾. Likewise, a more recent meta-analysis also showed that the beneficial effect of folic acid on stroke risk was to be found in trials longer than 3 years in duration, with a homocysteine-lowering effect of 20% or greater and in non-secondary prevention of stroke (Table 2)⁽Reference Lee, Hong and Chang¹⁹⁾. In addition the latter meta-analysis suggested for the first time that folic acid intervention might be more beneficial in men than in women; however, this finding requires further verification as none of the trials evaluated had exclusively investigated men or women. The evidence from these meta-analyses of RCT, together with the aforementioned North American population data suggesting a beneficial effect of increased folate status (through mandatory fortification) in preventing stroke, means that the case for enhanced folate and/or homocysteine-lowering in preventing CVD is stronger for stroke than for heart disease, and perhaps strongest for the primary prevention of stroke.

Table 1. Randomised controlled trials (RCT) of homocysteine-lowering and CVD events: secondary prevention

MI, myocardial infarction; CAD, coronary artery disease; TIA, transient ischaemic attack; RR, relative risk; HR, hazard ratio; FA, folic acid; B12, vitamin B₁₂; B6, vitamin B₆.

Table 2. Randomised controlled trials (RCT) of homocysteine-lowering and risk of stroke: a meta-analysis

N/a, not applicable.

* Referred to as secondary and non-secondary stroke prevention in Lee et al. meta-analysis⁽Reference Lee, Hong and Chang¹⁹⁾.

Evidence from genetic studies

Other evidence for a causal relationship between low folate status or elevated homocysteine and CVD comes from genetic studies. The most important genetic determinant of homocysteine in the general population is the common C677T variant in the gene encoding the folate-metabolising enzyme methylenetetrahydrofolate reductase (MTHFR) required for the formation of 5-methyltetrahydrofolate which, in turn is required in the remethylation of homocysteine to methionine. People who are homozygous for this polymorphism (TT genotype), have reduced MTHFR activity resulting in impaired folate metabolism and higher homocysteine concentrations in vivo ⁽Reference Frosst, Blom and Milos²⁰⁾; a phenotype that is found to be particularly marked in people who also have low status of folate⁽Reference Jacques, Bostom and Williams²¹⁾. The MTHFR 677 TT genotype has a reported frequency of 10% worldwide, ranging from 4 to 26% in Europe, 20% in Northern China to as high as 32% in Mexico⁽Reference Wilcken, Bamforth and Li²²⁾. Evidence from various meta-analyses of genetic studies including data for homocysteine concentrations and CVD events shows that individuals with the MTHFR 677 TT genotype have a significantly higher risk of CVD (by 14–21%) compared with those without this polymorphism⁽Reference Wald, Law and Morris^7, Reference Klerk, Verhoef and Clarke^23, Reference Lewis, Ebrahim and Davey-Smith²⁴⁾. The trend towards increasing risk of disease shown among individuals with wild-type (CC), heterozygous (CT) and homozygous mutant (TT) genotypes is consistent with the increasing gradient in homocysteine concentrations typically found among the three genotypes⁽Reference Wald, Law and Morris^7, Reference Klerk, Verhoef and Clarke²³⁾, as would be expected if there is a causal relationship between elevated homocysteine and risk of disease. Analysis of the OR among different countries, however, shows a large geographical variation in the extent of association between this polymorphism and CVD risk. Meta-analyses, which have examined this variation in relation to heart disease, show that the excess risk is not significant in North American populations, while the TT genotype carries a significantly increased (but variable) risk of CHD elsewhere in the world⁽Reference Klerk, Verhoef and Clarke^23, Reference Lewis, Ebrahim and Davey-Smith²⁴⁾. The largest and most recent investigation in this area, a collaboration of genetic studies consisting of data on almost 60 000 individuals including 20 885 stroke events, reported that the association between the MTHFR 677TT genotype and stroke was evident only in regions of low folate, whereas in regions with folic acid fortification policies the effect of MTHFR genotype was null⁽Reference Holmes, Newcombe and Hubacek²⁵⁾. Thus, the general conclusion emerging from genetic studies to date is one of effect modification by population dietary folate on the association between this polymorphism and CVD risk.

The mechanism whereby this genetic variant is linked with an increased risk of CVD is not generally dealt with in these reports but is generally assumed to relate to the phenotype of elevated plasma homocysteine in some way⁽Reference Klerk, Verhoef and Clarke^23–Reference Holmes, Newcombe and Hubacek²⁵⁾. Riboflavin, the co-factor for MTHFR, has been largely overlooked as a potential modulator of the phenotype, or indeed the cardiovascular risk, associated with the MTHFR C677T polymorphism. This omission is despite evidence that intervention with riboflavin lowers plasma homocysteine specifically in individuals with the MTHFR 677TT genotype, and unlike folate and vitamin B₁₂, riboflavin does not have a homocysteine-lowering effect in the other MTHFR genotype groups⁽Reference McNulty, Dowey le and Strain⁴⁾. This finding suggests that riboflavin intervention can in some way stabilise MTHFR activity in individuals with the MTHFR 677TT genotype and restore enzyme activity to levels found in those without this genotype.

Folate

Taken together, the evidence showing a decline in stroke-related mortality in North America corresponding to the introduction of mandatory folic acid-fortification⁽Reference Yang, Botto and Erickson⁸⁾, along with the evidence from RCT showing beneficial effects of folic acid in people with no previous history of stroke⁽Reference Wang, Qin and Demirtas^18, Reference Lee, Hong and Chang¹⁹⁾, suggest that folic acid may have an important role to play in the primary prevention of stroke. Thus, although aimed at preventing neural-tube defects, folic acid fortification on a population-wide basis may also prevent stroke. Randomised trials that have failed to show beneficial effects of folic acid on stroke have in general been conducted in countries with a high prevailing folate status⁽Reference Holmes, Newcombe and Hubacek²⁵⁾. Thus, it is unlikely that further increases in folate intake will be effective in a population whose folate status is generally optimal. However, while overt folate deficiency (i.e. megaloblastic anaemia) is relatively rare in the absence of an underlying clinical cause, many otherwise healthy populations have inadequate folate intakes when considered from the perspective of achieving optimal status rather than merely preventing deficiency of folate⁽Reference McNulty and Scott³²⁾. The poor stability and poor bioavailability of folate from natural food sources compared with folic acid (the synthetic vitamin form found in fortified foods and supplements) means that achieving optimal folate status in a population in the absence of a folic acid fortification policy can be a major challenge⁽Reference McNulty and Scott³²⁾. Thus, in populations with limited or no access to fortified foods where consumers are dependent on natural food folate sources to meet their dietary intakes, folate status could be predicted to be low⁽Reference Hoey, McNulty and Askin³³⁾. Intervention could be anticipated to have the greatest benefit in reducing the risk of stroke in these regions, and it is suggested that any future RCT of homocysteine-lowering intervention for stroke prevention should focus on such populations with low prevailing folate intake and status⁽Reference Holmes, Newcombe and Hubacek²⁵⁾.

Balanced against any potential benefits of folic acid in preventing stroke, however, are concerns regarding potential adverse effects of long-term exposure to high folic acid levels. Thus, mandatory folic acid fortification (aimed at preventing neural-tube defects) remains very controversial, despite the relative ineffectiveness of alternative strategies to increase folate status through health promotion campaigns⁽Reference McNulty, Pentieva and Marshall³⁴⁾. The major concern traditionally focused on the potential risk that high folic acid intake might mask the anaemia of vitamin B₁₂ deficiency⁽Reference Reynolds³⁵⁾, but more recently the concern emerged that it could be associated with an increased risk of cognitive dysfunction in older people with low-vitamin B₁₂ status⁽Reference Morris, Jacques and Rosenberg³⁶⁾. Furthermore, despite considerable evidence that higher folate within the dietary range plays a protective role against cancer at various sites⁽Reference Kim^37–Reference Gibson, Weinstein and Pfeiffer³⁹⁾, other evidence suggests that exposure to high-dose folic acid (>1 mg/d) may promote colorectal tumorigenesis in patients with pre-existing lesions⁽Reference Cole, Baron and Sandler⁴⁰⁾ or even increase cancer risk generally⁽Reference Figueiredo, Grau and Haile^41, Reference Ebbing, Bønaa and Nygård⁴²⁾. Thus, some remain concerned that excessively high folic acid intakes could have a cancer-promoting effect in segments of aging populations⁽Reference Mason⁴³⁾, and policy-makers worldwide (including the UK government) have delayed decisions to implement population-based folic acid fortification policies similar to those introduced in 1998 in North America.

Should emerging evidence prove the role of folic acid in the primary prevention of stroke, then the public health question of setting the appropriate folic acid dose to achieve this beneficial effect in a population via food fortification will need to be answered. A lower target dose for beneficial effects will lower the risk of adverse effects (whatever the estimated size of such risk) in any emerging fortification policy. One recent trial from our Centre addressed the issue of determining the lowest dose of folic acid required to achieve a maximal lowering in plasma homocysteine in heart disease patients and age- and sex-matched controls⁽Reference Tighe, Ward and McNulty⁴⁴⁾. The results showed that a dose of folic acid as low as 0·2 mg/d can, if given chronically, maximally lower homocysteine in patients and controls; higher doses (0·4 and 0·8 mg/d) resulted in no additional lowering. By sampling participants at both 6 and 12 weeks, in addition to the full intervention period of 26 weeks, the trial also indicated that the majority of previous studies used too short an intervention period to observe the full extent of homocysteine-lowering in response to low folic acid doses and thus concluded that the much higher folic acid dose of 0·8 mg/d was required⁽¹⁾. Thus, the potency of folic acid at low doses given chronically (as in food fortification) was previously underestimated, and these findings indicate that long-term exposure to high-dose folic acid through food fortification is unnecessary⁽Reference Tighe, Ward and McNulty⁴⁴⁾.

Vitamin B₁₂

Vitamin B₁₂, like folate, is required for the remethylation of homocysteine to methionine (Fig. 1). Thus, achieving optimal folate status alone may not lower homocysteine to desirable concentrations (<10 μmol/l)⁽Reference Malinow, Bostom and Krauss⁴⁵⁾, and optimising vitamin B₁₂ may have benefits over and above the effect of folate alone. A further 7% reduction in homocysteine (additional to that with folic acid alone) can be achieved with vitamin B₁₂⁽Reference Hoey, Strain and McNulty⁴⁶⁾. Any small additional decrease in homocysteine could be predicted to confer a further benefit in terms of cardiovascular risk given that the relationship with homocysteine is a graded one^(6, Reference Wald, Law and Morris⁷⁾. Although vitamin B₁₂ intakes are generally found to be more than adequate in most healthy populations (often greatly exceeding recommended values), the achievement of an optimal status may be greatly hindered for many older people because of the common problem of age-related food-bound vitamin B₁₂ malabsorption, estimated to affect up to 40% of otherwise healthy older people⁽Reference Clarke, Refsum and Birks^47, Reference Carmel⁴⁸⁾. This problem arises mainly from atrophic gastritis, a chronic inflammatory condition, resulting in decreased gastric acid production (hypochlorhydria), which diminishes vitamin B₁₂ absorption because of the essential role of gastric acid in the release of protein-bound vitamin B₁₂ from food⁽Reference Carmel⁴⁸⁾. Unlike the treatment required for the classical (but rare) vitamin B₁₂ deficiency sign of pernicious anaemia requiring vitamin B₁₂ injections for life, older adults with low vitamin B₁₂ status owing to food-bound malabsorption should be able to absorb free (crystalline) vitamin B₁₂ because it is not bound to protein. In fact, on the basis of this assumption, the Institute of Medicine in the USA recommends that people aged 50 years and over consume most of their vitamin B₁₂ from crystalline vitamin B₁₂ found in fortified foods and supplements⁽⁴⁹⁾. No such recommendation exists in the UK or any European country.

Riboflavin

Riboflavin is required as a co-factor for the folate-metabolising enzyme MTHFR and therefore is needed to generate 5-methyltetrahydrofolate which, in turn, acts in the remethylation of homocysteine to methionine (Fig. 1), but the potential role of riboflavin as a C₁ donor is often overlooked. The emerging evidence reviewed here that the higher blood pressure found in patients with a common genetic variant in MTHFR is highly responsive to low-dose riboflavin⁽Reference Horigan, McNulty and Ward²⁹⁾, raises the possibility that improving riboflavin status will have an important role in preventing hypertension and therefore in modulating the risk of stroke specifically in the 10% of people worldwide (and much higher in certain populations) with the relevant genotype⁽Reference Wilcken, Bamforth and Li²²⁾.

The Scientific Advisory Committee on Nutrition, however, has expressed concern about the high proportion of the British adult population with apparently poor riboflavin status as determined from national survey data using the gold standard biomarker of status, erythrocyte glutathione activation coefficient⁽Reference Ruston, Hoare and Henderson⁵⁰⁾. In general, dietary intakes (provided in the diet predominantly through consuming milk and dairy foods) were found to compare favourably with recommended values; however, high proportions of young women were found to have low intakes⁽Reference Ruston, Hoare and Henderson⁵⁰⁾. Thus, it is unclear at this time as to whether or not the large proportion of the UK population with abnormal erythrocyte glutathione activation coefficient values indicates a generalised problem of poor riboflavin status; this requires further investigation.