Elevated homocysteine as a risk factor for CVD
Evidence from numerous prospective and retrospective case–control studies has emerged in recent years to link elevated plasma homocysteine (tHcy) levels with an increased risk of CVD. Meta-analyses of prospective studies have predicted that lowering tHcy by 3 μmol/l (or a reduction of 25% based on an average tHcy of 12 μmol/l) would reduce the risk of heart disease by 11–16% and stroke by 19–24%(1, Reference Wald, Law and Morris2). Although none of the secondary prevention trials published in more recent years have been able to confirm the benefit of tHcy-lowering therapy on CVD events generally(Reference Toole, Malinow, Chambless, Spence, Pettigrew, Howard, Sides, Wang and Stampfer3–Reference Bonaa, Njølstad and Ueland5), it should not be assumed that no relationship exists. It is now generally recognised that these trials lacked sufficient statistical power to detect an effect for the predicted magnitude of association between tHcy and heart disease(6). In support of this viewpoint, a clear benefit in reducing stroke was shown in one of the previously mentioned ‘negative’ trials, although this result was not explicit in the conclusions(4). Moreover, evidence just published from a meta-analysis of clinical trials in relation to stroke outcome has confirmed that folic acid supplementation reduces the risk of stroke by 18% overall, by 29% in trials with a treatment duration of >36 months and by 25% in those trials involving individuals without a history of stroke(Reference Wang, Qin, Demirtas, Li, Mao, Huo, Sun, Liu and Xu7). Such evidence is consistent with the findings of a population-based study that has shown that the temporal decline in stroke-related mortality in the USA and Canada coincided with the introduction of folic acid fortification(Reference Yang, Botto, Erickson, Berry, Sambell, Johansen and Friedman8). Thus, the case for tHcy as a risk factor in CVD is stronger for stroke than for heart disease, and possibly strongest for the primary prevention of stroke.
Another important line of evidence supporting a causal relationship between elevated tHcy and heart disease comes from genetic studies. The common C677T variant in the gene coding for the folate-metabolising enzyme methylenetetrahydrofolate reductase (MTHFR) is the most important genetic determinant of tHcy in the general population. The homozygous mutant TT genotype for the MTHFR C677T polymorphism typically affects about 10% of individuals worldwide but can be as high as 26% (southern Italy) and 32% (Mexico) in some areas(Reference Wilcken, Bamforth and Li9). MTHFR is required for the formation of 5-methyltetrahydrofolate, which in turn is required to convert homocysteine to methionine. Individuals with the TT genotype have reduced MTHFR activity, which results in impaired folate metabolism and elevated tHcy levels(Reference Frosst, Blom and Milos10). Three recent meta-analyses involving >25 000 cases have examined the impact on heart disease risk of this genetic variant and have shown an overall significantly higher (14–21%) risk of heart disease in individuals with this polymorphism as compared with those without this polymorphism(Reference Wald, Law and Morris2, Reference Klerk, Verhoef, Clarke, Blom, Kok and Schouten11, Reference Lewis, Ebrahim and Davey Smith12). The trend toward increasing risk of disease among individuals with no (CC genotype), one (CT genotype) or two copies (TT genotype) of the defective gene is consistent with the increasing gradient in tHcy typically found among the three genotypes (as would be expected if there is a causal relationship between elevated tHcy and risk of disease). However, analysis of the OR among countries shows a large geographical variation in the association between this polymorphism and heart disease risk(Reference Klerk, Verhoef, Clarke, Blom, Kok and Schouten11, Reference Lewis, Ebrahim and Davey Smith12).
Homocysteine-lowering with B-vitamins
The B-vitamins folate, vitamin B12 and vitamin B6 are required for homocysteine metabolism and several studies have examined their potential to lower tHcy.
Folate, the focus of much current debate on food fortification worldwide, is the major determinant of tHcy. The first meta-analysis to assess the tHcy-lowering effect of folic acid has concluded that doses in the range of 0·5 to 5 mg/d could lower tHcy by about 20–25%(13). The effect of doses <0·5 mg/d is much less clear but is arguably the more relevant question for emerging food fortification policy. Some earlier evidence has suggested that doses in the range of 0·2–0·4 mg/d can achieve a maximum reduction in tHcy in young healthy populations(Reference Ward, McNulty, McPartlin, Strain, Weir and Scott14, Reference Daly, Mills, Molloy, Conley, McPartlin, Lee, Young, Kirke, Weir and Scott15), while another study has shown that doses as high as 0·8 mg folic acid/d are required for maximal lowering of tHcy in those individuals with established heart disease(Reference Wald, Bishop, Wald, Law, Hennessy, Weir, McPartlin and Scott16). Similarly, a meta-analysis of twenty-five randomised trials involving 2596 subjects (both healthy and patient groups) has concluded that a daily dose of 0·8 mg/d is required to achieve a maximal reduction in tHcy, while doses of 0·2 and 0·4 mg/d are associated with only 60 and 90% of this maximal effect respectively(17). However, one limitation of the latter meta-analysis is treatment duration; of the twenty-five studies examined the treatment in eighteen was of ⩽8 weeks duration, six were of 12 weeks duration and only one was of long-term duration (i.e. 6 months). Some preliminary evidence suggests that longer treatment duration may be necessary to allow a complete tHcy-lowering effect to be observed in response to the lower doses of folic acid(Reference Tighe, Ward, McNulty, Finnegan, Strain, Dunne, Molloy and Scott18). In addition, details on subject compliance were rarely reported in the original studies that examined the effect of different doses(17), but this information is critical to the interpretation of the results because incomplete compliance by participants may result in an underestimation of the full extent of the tHcy response to a given dose. Food fortification, unlike supplementation, automatically provides maximal participant compliance; therefore, ensuring high subject compliance to the treatments should be a key aspect of any dose-finding study aimed at developing food fortification policies for the prevention of CVD.
Thus, some published reports may have overestimated the folic acid dose required to achieve maximal tHcy lowering, because of either incomplete or unmonitored subject compliance or, more importantly, an intervention period that was too short to observe the full extent of the tHcy response to lower folic acid doses in the range 0·2–0·4 mg/d. Clearly, the issue as to the lowest dose of folic acid associated with a maximum reduction in tHcy remains controversial and requires further investigation, especially in light of recent reports that highlight potential adverse effects of overexposure to higher intakes of folic acid(Reference Troen, Mitchell and Sorensen19, Reference Cole, Baron and Sandler20).
In addition to folate, the enzyme methionine synthase involved in the remethylation of homocysteine to methionine requires vitamin B12 as cofactor. Although a number of studies in healthy populations and in patients have investigated the effect of various doses of folic acid supplementation in lowering tHcy, few have examined the independent effects of vitamin B12. In those studies that have administered low-dose vitamin B12 as a separate treatment, the response of vitamin B12 biomarkers was the primary outcome and the tHcy response was measured as a secondary objective(Reference Seal, Metz, Flicker and Melny21, Reference Eussen, De Groot, Clarke, Schneede, Ueland, Hoefnagels and van Staveren22). Furthermore, the study populations were selected for mild vitamin B12 deficiency and were not pretreated with folic acid before vitamin B12 supplementation. Of even greater importance, vitamin B12 was not administered with food, the presence of which is required to stimulate the normal vitamin B12 absorptive mechanisms. The tHcy-lowering response observed may therefore have been confounded by differences in folate status or an incomplete absorption of the administered dose.
The reason for the limited number of intervention studies with vitamin B12 is that this vitamin is generally considered to be a far-less-effective determinant of tHcy concentrations compared with folate. However, evidence from a study of healthy subjects supplemented with low-dose folic acid(Reference Quinlivan, McPartlin, McNulty, Ward, Strain, Weir and Scott23) and from studies in the era of mandatory folic acid fortification of cereal grains in the USA(Reference Liaugaudas, Jacques, Selhub, Rosenberg and Boston24–Reference Robertson, Lemolo, Stabler, Allen and Spence26) shows that vitamin B12 becomes the main nutritional determinant of tHcy once folate status is optimised. Furthermore, meta-analyses of intervention studies that have examined the effect of B-vitamins on tHcy lowering have shown that including vitamin B12 along with folate produces an additional one-third (7%) lowering of tHcy above that achieved with folate alone (typically 25% lowering(13, 17)). As the relationship between tHcy and CVD is graded, any small but significant further decrease in tHcy concentrations could be predicted to confer an additional benefit in terms of CVD risk. Although the results of the randomised clinical trials published so far are disappointing in that they have failed to show a benefit of tHcy-lowering therapy on CVD events generally, re-analysis of data from the negative Vitamin Intervention for Stroke Prevention trial(Reference Toole, Malinow, Chambless, Spence, Pettigrew, Howard, Sides, Wang and Stampfer3) has shown the importance of vitamin B12 in relation to tHcy and CVD risk. When subjects who were considered to receive no benefit from vitamin therapy were excluded (i.e. patients believed to be on vitamin B12 supplements and patients with renal failure), the group receiving the high-dose therapy were found to have a 21% reduced risk of CVD events compared with the low-dose group(Reference Spence, Bang, Chambless and Stampfer27). Those patients with higher baseline vitamin B12 levels who received the high-dose B-vitamin therapy during intervention were shown to have the best outcome for survival free of a CVD event(Reference Spence, Bang, Chambless and Stampfer27). Thus, the inclusion of vitamin B12 along with folic acid may be a more effective therapy for reducing the risk of CVD via tHcy lowering than folic acid alone. Such a therapy could be of particular benefit to older adults, as suboptimal vitamin B12 status (mostly as a result of food-bound vitamin B12 malabsorption) is highly prevalent(Reference Wolters, Strohle and Hahn28) and CVD is a common cause of morbidity and mortality in this subgroup of the population. However, further supplementation studies with vitamin B12 are required to determine the optimal dose for lowering tHcy.
The transulfuration of homocysteine to cysteine by cystathione β-synthase requires vitamin B6 (pyridoxal phosphate) as a cofactor, and studies have considered the potential of vitamin B6 to lower tHcy. Although the literature generally suggests either that vitamin B6 does not have a tHcy-lowering effect(13, 17) or that it lowers tHcy only in exceptional circumstances (e.g. in pyridoxine-responsive homocystinuria or in patients with severe vitamin B6 deficiency), there is some evidence of a small but significant tHcy response (lowered by 7·5%) to low-dose vitamin B6(Reference McKinley, McNulty, McPartlin, Strain, Pentieva, Ward, Weir and Scott29). However, the latter intervention was conducted in healthy older subjects whose status of both folate and riboflavin was optimised before intervention with vitamin B6; this study design may explain why a significant tHcy response was observed in this study but not in other studies(13, 17).
Apart from its role in homocysteine metabolism, vitamin B6 is also required for the metabolism of n-3 PUFA(Reference Tsuge, Hotta and Hayakawa30). Thus, independently of any tHcy-lowering effect, some studies have investigated the potential ability of vitamin B6 to suppress some of the underlying mechanisms involved in the atherosclerotic process such as platelet aggregation(Reference Chang, Chang and Chen31) and the proliferation of endothelial cells(Reference Matsubara, Matsumoto, Mizuchina, Lee and Kato32). The findings so far from various studies are inconsistent. The results from the Nurses' Health Study of 80 000 women followed for 14 years have demonstrated that a higher dietary intake of vitamin B6 (i.e. >3 mg/d) is associated with a decreased risk of heart disease(Reference Rimm, Willett, Hu, Sampson, Colditz, Manson, Hennekens and Stampfer33). Furthermore, retrospective and prospective case–control studies have related low plasma concentrations of vitamin B6-status indices to increased risk of stroke, peripheral vascular disease and coronary artery disease(Reference Chasan-Taber, Selhub, Rosenberg, Malinow, Terry, Tishler, Willett, Hennekens and Stampher34–Reference Kelly, Shih, Kistler, Barron, Lee, Mandell and Furie37). However, after adjusting for established CVD risk factors the relationship between vitamin B6 indices and CVD in some of these studies was found to be no longer significant(Reference Chasan-Taber, Selhub, Rosenberg, Malinow, Terry, Tishler, Willett, Hennekens and Stampher34, Reference Kelly, Kistler, Shih, Mandell, Atassi, Barron, Lee, Silveira and Furie38). More-recent investigations have reported that low vitamin B6 status is related to chronic inflammation(Reference Friso, Jacques, Wilson, Rosenberg and Selhub39), which in turn is known to promote atherosclerosis and the development of CVD. This finding might suggest that low vitamin B6 status is not an independent risk factor for CVD and stroke but merely a marker of chronic inflammation. Evidence as to whether low vitamin B6 status is a causal factor for CVD can only be provided by controlled intervention studies. So far, the results of four randomised controlled trials that included vitamin B6 supplementation (as an independent treatment or in combination with folic acid and vitamin B12) for ⩽5 years in patients with pre-existing CVD have been published, but they have failed to detect any impact of vitamin B6 on the risk of a recurrent cardiovascular event(Reference Clarke, Lewington, Sherliker and Armitage40). However, it is widely acknowledged that these trials were underpowered, and even after combining them it appears that there is insufficient power to detect a significant effect on the risk of CVD(Reference Clarke, Lewington, Sherliker and Armitage40). Moreover, the doses of vitamin B6 used in these trials were pharmacological levels (40 mg/d), the long-term effects of which remain unknown. Finally, these trials were conducted in individuals with pre-existing disease and it is not known whether vitamin B6 would have a role in the primary prevention of CVD. Carefully-designed and sufficiently-powered long-term intervention studies examining the effect on CVD of low doses of vitamin B6 (within the dietary range) are clearly warranted.
The typical phenotype associated with homozygosity for the MTHFR C677T polymorphism is elevated tHcy levels(Reference Frosst, Blom and Milos10). Individuals with the TT genotype are considered to have increased dietary folate requirements on the basis that they have lower erythrocyte folate levels compared with those without this genetic variant(Reference Molloy, Daly, Mills, Kirke, Whitehead, Ramsbottom, Conley, Weir and Scott41), and the increase in tHcy is found to be most marked among those with lower folate status(Reference Jacques, Bostom, Williams, Ellison, Eckfeldt, Rosenberg, Selhub and Rozen42, Reference Brattström, Wilcken, Ohrvik and Brudin43).
A fourth, much overlooked, B-vitamin is also involved in homocysteine metabolism. In addition to folate, riboflavin (in its co-enzymic form FAD) is required as a cofactor for the MTHFR enzyme. The reduced activity of the MTHFR 677TT variant of MTHFR has been shown to result from the inappropriate loss of its FAD cofactor(Reference Yamada, Chen, Rozen and Matthews44). Observational studies in human subjects that have investigated the relationship between riboflavin status and tHcy in individuals with different MTHFR genotypes have reported somewhat inconsistent results(Reference Jacques, Kalmbach, Bagley, Russo, Rogers, Wilson, Rosenberg and Selhub45–Reference McNulty, McKinley, Wilson, McPartlin, Strain, Weir and Scott47). In the USA an association between riboflavin status and tHcy has been reported in individuals with the TT genotype in the Framingham Cohort(Reference Jacques, Kalmbach, Bagley, Russo, Rogers, Wilson, Rosenberg and Selhub45), but this association is confined only to those with the TT genotype and low folate status; thus, riboflavin does not seem to be the limiting nutrient. The two studies conducted in healthy European populations (Norway and Northern Ireland), however, have both identified riboflavin as an important determinant of tHcy among individuals with the TT genotype and have indicated that this effect is not explained by folate(Reference Hustad, Ueland, Vollset, Zhang, Bjorke-Monsen and Schneede46, Reference McNulty, McKinley, Wilson, McPartlin, Strain, Weir and Scott47). The inconsistency between studies is probably explained by the mandatory fortification of flour with riboflavin in the USA, which would have the effect of optimising riboflavin status in the general US population, thereby reducing the extent to which riboflavin is found to be a limiting nutrient in determining tHcy levels in individuals with the TT genotype. Recent results that show a genotype-specific response of tHcy to riboflavin supplementation now confirm that riboflavin is an independent modifier of tHcy in individuals with the TT genotype. Significant lowering of tHcy in response to riboflavin supplementation was observed in healthy individuals with the TT genotype, with levels decreasing by as much as 22% overall, and markedly so (by 40%) in those with lower riboflavin status at baseline(Reference McNulty, Dowey, Strain, Dunne, Ward, Molloy, McAnena, Hughes, Hannon-Fletcher and Scott48). No tHcy response to intervention was observed in those with CC or CT genotypes, despite a significant improvement in riboflavin status in both cases and the pre-selection of subjects with suboptimal riboflavin status at baseline. The lack of a tHcy-lowering effect of riboflavin in the absence of this polymorphism was observed previously, in an earlier intervention study of healthy elderly individuals who were also pre-screened for suboptimal riboflavin status but not for MTHFR genotype(Reference McKinley, McNulty, McPartlin, Strain and Scott49). The responsiveness of tHcy to riboflavin is therefore specific to individuals with the MTHFR 677TT genotype and represents a new gene–nutrient interaction.
The inconsistencies in the literature as to whether the MTHFR C677T polymorphism is associated with a higher risk of heart disease may also relate to the role of riboflavin. Although meta-analyses have reported an overall 14–21% higher risk of heart disease in individuals with the TT genotype(Reference Wald, Law and Morris2, Reference Klerk, Verhoef, Clarke, Blom, Kok and Schouten11, Reference Lewis, Ebrahim and Davey Smith12), analysis of the OR between continents shows that the excess heart disease risk associated with this polymorphism is significant in Europe but not in North America. This geographical variation is generally assumed to be the result of differences in folate status(Reference Klerk, Verhoef, Clarke, Blom, Kok and Schouten11, Reference Lewis, Ebrahim and Davey Smith12), while riboflavin, the cofactor for MTHFR, is generally overlooked as a potential modulator of the disease risk associated with this polymorphism. However, the policy of mandatory riboflavin fortification has existed for >50 years in the USA, and given the marked genotype-specific decrease in tHcy with riboflavin intervention(Reference McNulty, Dowey, Strain, Dunne, Ward, Molloy, McAnena, Hughes, Hannon-Fletcher and Scott48), it is not unreasonable to suggest that it might also modulate heart disease risk in this subpopulation with genetic predisposition to elevated tHcy. Thus, the reported differences among countries as to whether this polymorphism represents an increased risk of heart disease may relate not only to differences in folate as commonly suggested(Reference Klerk, Verhoef, Clarke, Blom, Kok and Schouten11, Reference Lewis, Ebrahim and Davey Smith12), but also to differences in the prevailing riboflavin status. It could be predicted that individuals with the TT genotype who also have low riboflavin status would have an excess risk of heart disease, whereas with optimal riboflavin status they would not carry the expected risk. Notably, support for this viewpoint is offered by very recent evidence of higher blood pressure at baseline and a marked lowering of both systolic and diastolic blood pressure in response to riboflavin intervention specifically in patients with the TT genotype(Reference Horigan, Ward, McNulty, Purvis, Strain and Scott50).
Implications for nutrition policy
Folate is the major determinant of tHcy. As has been experienced in the USA since its introduction(Reference Jacques, Selhub, Bostom, Wilson and Rosenberg51), there is no doubt that folic acid fortification, if introduced in a population, would have a major tHcy-lowering effect irrespective of genotype. Vitamins B12 and B6 also play key roles in homocysteine metabolism, with evidence showing that both vitamins may be important determinants of tHcy, particularly when folate status has been optimised(Reference Quinlivan, McPartlin, McNulty, Ward, Strain, Weir and Scott23, Reference Liaugaudas, Jacques, Selhub, Rosenberg and Boston24, Reference McKinley, McNulty, McPartlin, Strain, Pentieva, Ward, Weir and Scott29). Thus, all three vitamins could arguably be included in any fortification policy aimed at lowering tHcy in the general population, with potential benefits in preventing CVD and possibly stroke in particular(Reference Wang, Qin, Demirtas, Li, Mao, Huo, Sun, Liu and Xu7). Emerging food policy in different countries should also consider the riboflavin requirements of individuals homozygous for the common MTHFR C677T polymorphism. Riboflavin status appears to be a potent modulator of the expected (high tHcy) phenotype among individuals with the TT genotype. Of greater importance, new evidence shows marked lowering of blood pressure in response to riboflavin intervention specifically in patients with the TT genotype, which may or may not be independent of the tHcy-lowering effect of riboflavin also seen only in the TT genotype(Reference McNulty, Dowey, Strain, Dunne, Ward, Molloy, McAnena, Hughes, Hannon-Fletcher and Scott48, Reference Horigan, Ward, McNulty, Purvis, Strain and Scott50). Importantly, the fact that these effects of riboflavin are achievable with a very modest increase in riboflavin intake (1·6 mg/d) suggests that there are important implications for dietary riboflavin requirements in individuals with this common genetic variant and for food-fortification policy aimed at the primary prevention of CVD. In order to cover the needs of appreciable subgroups (3–32%) of populations worldwide with this genotype(Reference Wang, Qin, Demirtas, Li, Mao, Huo, Sun, Liu and Xu7) riboflavin may need to be considered for inclusion together with folic acid in fortification programmes under discussion.