Homocysteine (Hcy) is a non-essential, non-protein-forming amino acid that originates in methionine via a transmethylation reaction. Hcy is catabolized into methionine or cysteine. Methionine synthase and its co-factor, methylcobalamin, enhance the methylation of Hcy into methionine, where 5-methyl tetrahydrofolate donates its methyl group to Hcy. The trans-sulfuration pathway of Hcy catabolism is mediated via cystathionine beta synthase (CBS) and cystathionase, both of which are vitamin-B6-dependent. The most common cause of moderate hyperhomocysteinemia ([HHCY] >12–30μmol/l) is related to a deficiency of one or more of the B vitamins (folate, vitamin B12, or vitamin B6).1–3 Genetic polymorphisms (e.g. methylenetetrahydrofolate reductase deficiency [MTHFR]) or renal insufficiency4 can also cause moderate HHCY.
Early investigations on patients with a CBS deficiency have suggested that severe HHCY is a risk factor for atherogenic and atherothrombotic diseases. Elevated concentrations of plasma total Hcy (tHcy >12μmol/l), which is common in elderly people, is regarded as an independent risk factor for cardiovascular disease.5 The prevalence of moderate HHCY is high in elderly patients (30–50% in patients above 65 years of age). Elderly patients often suffer from diseases associated with HHCY. Thus, a lowering of the tHcy level by a vitamin B treatment seems to present a promising, low-priced, and simple preventive and therapeutic measure. Treating patients with homocysteinuria with vitamin B6 and/or folate has proved meaningful for improving cardiovascular outcome. The same approach in moderate HHCY has gained interest in recent years. Numerous trials have begun with the aim of testing the hypothesis that lowering tHcy will decrease the risk for coronary vascular disease (CVD). Several trials have been published during the last five years, but the hypothesis remains obscure and the new results have only complicated the main hypothesis. However, recent data from a meta-analysis of treatment studies on the lowering of stroke risk6 confirmed the effectiveness of tHcy lowering by B vitamins and contributed to a clarification of existing contradictions (see Figure 1). This article seeks to contribute to a scientific evaluation of the significance of lowering tHcy by B vitamins and to evaluate the relevance of the latter.
Causal Correlation Between Hyperhomocysteinemia and Cardiovascular Disease
Large meta-analyses7,8 of retrospective and prospective studies stress the causal correlation between HHCY and degenerative (vascular) diseases. The results imply that each 5μmol/l increase in plasma tHcy is associated with an odds ratio (OR) of 1.32 for ischemic heart disease, 1.60 for thrombosis, and 1.59 for stroke. Furthermore, these meta-analyses suggested that a lowering of tHcy concentration by 3μmol/l might reduce the risk for ischemic heart disease by 16%, for thrombosis by 25%, and for stroke by 24%.8
Can Treating Hyperhomocysteinemia Provide Protection Against Stroke and Coronary Vascular Disease?
One prospective study that lasted for more than four years observed a significant reduction of plaque within the carotid artery9 owing to B-vitamin supplementation. Moreover, a significant reduction of the carotid intima-media thickness in patients at risk for stroke has recently been reported after treatment with B vitamins lasting for one year.10 However, several recent treatment studies could not detect a benefit for B-vitamin supplementation, thus leaving open questions about the repeatedly observed association between HHCY and vascular risk in retrospective and prospective studies. Notably, available trials have inherent errors and are not optimized to address the role of tHcy lowering on primary or secondary prevention of CVD.
It is critically important to review the limitations of the available studies before reaching a firm conclusion about the protective effect of the B vitamins against CVD and stroke. Table 1 lists factors to be considered when planning or interpreting studies on the effect of Hcy-lowering treatment on the risk for stroke or coronary vascular diseases. One serious problem of available studies is that instead of comparing non-treated with treated patients, the results of conventional treatment are compared with the results of conventional treatment to which vitamins are added. Most of these studies included patients with several medications that interact with tHcy metabolism or levels. Worldwide, more than 50,000 patients are currently included in intervention studies in order to determine the possible benefits of vitamin therapy (secondary prevention). First intervention studies, such as the Vitamin Intervention for Stroke Prevention (VISP),11 Norwegian Vitamin Trial (NORVIT),12 Heart Outcomes Prevention Evaluation-2 Study (HOPE 2),13 Women’s Antioxidant and Folic Acid Cardiovascular Study (WAFACS),14 and Western Norway B-vitamin Intervention Trial (WENBIT)15 studies, have been completed and published.
The NORVIT study included 3,749 patients who had suffered a myocardial infarction at most seven days before inclusion in the study. The patients were treated with B vitamins (divided into four therapeutic groups or placebo in a two-by-two factorial design) in addition to conventional medication for three years. The tHcy level was lowered significantly—by 28% in the group that received folic acid and vitamin B12. There was no risk reduction regarding the end-points (heart attack, stroke). This study did not eliminate numerous possible co-variables that may affect the end-points and potentially mask any therapeutic effect (stroke and myocardial infarction). Obviously, looking at the Kaplan-Meier estimates another weak point of this study is that half of the primary end-points occurred in the first half-year of treatment. Thus, patients with coronary events occurring shortly before the beginning of the study are not to be taken into account for the outcome of the study.
The VISP study included 3,860 stroke patients who were treated with conventional medication over two years and, additionally, in times of fortification with ‘low or high dosages’ of B vitamins (folic acid: low 200μg, high 2,500μg; vitamin B12: low 6μg, high 400μg; vitamin B6: low 0.2mg, high 25mg). The tHcy level decreased by only 2μmol/l in the high-dose therapy group. There was no significant effect on the end-points (stroke, coronary episodes, or death), even though there was a significant link between the baseline tHcy level and these end-points. Possible reasons for the lack of therapeutic effect are folate enrichment in grain products in the US during the study, the short observation period, and the fact that vitamin B12 status and kidney function were not taken into consideration.
The HOPE study included 5,222 patients with vascular disease or diabetes who were treated with B vitamins or placebo over a period of five years. In this study, the Hcy plasma level was lowered by 26%. As a result, it was concluded that a treatment combining folic acid, vitamin B6, and vitamin B12 did not result in a reduction in the number of severe cardiovascular events in patients with vascular diseases. Moreover, tHcy levels were assessed in only 581 treated patients and 588 controls in a consecutive manner, limiting the analysis of treatment on tHcy to approximately one-fifth of the cohort; therefore, any effect of the B vitamins on tHcy was seen only in these patients. Furthermore, an increased tHcy concentration was not one of the criteria for inclusion into the study, and neither a folic acid deficiency nor a deficiency of vitamin B12 or vitamin B6 was present in the patients. However, despite these limitations, a 5% lowering of risk by B-vitamin therapy was reported. Furthermore, a sub-group analysis of the HOPE 2 study revealed that vitamin treatment reduced the risk for stroke by about 25% (145 patients in the vitamin group and 111 patients in the placebo group).
A recent report from the HOPE-2 study documented that daily supplement of folic acid, vitamin B6, and vitamin B12 for five years reduced the risk for stroke by 25%.16 The beneficial effect of the vitamins was larger in subjects with certain risk profiles or medications and no marked effect can be expected in the first three years of supplementation. Patients receiving antiplatelet agent or cholesterol-lowering drugs or those coming from a country with folate fortification were less likely to benefit.16 The HOPE-2 study clearly showed that several confounding factors were underestimated in similar studies. If cholesterol lowering reduces the anticipated effect of tHcy lowering, the power calculation of numerous large trials must be reconsidered. As the protective effect of tHcy lowering was more impressive in the group with high tHcy and cholesterol, this suggests that HHCY can accelerate atherosclerosis when associated with elevated plasma lipids.
The newly published WAFACS study was a placebo-controlled, double-blind, vitamin-supplementation trial with folic acid 2.5mg plus vitamin B12 1mg and vitamin B6 50mg in 5,442 women at risk for CVD.14 The patients were participating in a risk reduction trial by antioxidant vitamins (vitamin C, vitamin E, and beta carotene). After 7.3 years of treatment, B vitamins did not reduce a combined end-point of total CVD events among high-risk women, despite a significant lowering of tHcy levels. There were several concerns regarding the study design and the validity of the findings. Approximately 64% of the women included in this study had prior CVD. For this main sub-group, the treatment aimed at prevention of future events. For the remaining participants (women at risk for CVD), the trial aimed at primary prevention of CVD. These two groups should be considered separately with regard to prevention. Furthermore, the criteria used to predict the risk for CVD are obscure and not in accordance with generally accepted criteria. Thus, the possibility that the participants in the WAFACS study with at least three risk factors were misclassified has to be considered as a candidate variable that might inflate the error.
Approximately 21.5% of the women were below 54 years of age, suggesting that the risk profile in younger women is different from that in elderly women (approximately 42.6% were 64 years of age). Menopausal status and hormone replacement treatment are important co-variables that should be taken into account. Cholesterol-lowering drugs lower the risk for future CVD and stroke. The criteria used to stratify people as having elevated cholesterol raise many concerns. As 64% of the patients already had CVD, the definition of hypercholesterolemia for this subgroup should depend on stricter cut-off values for total and low-density lipoprotein (LDL) cholesterol. Moreover, no information has been shown on the dose and type of treatment. On the one hand, despite the fact that 64% of the WAFACS study population had already experienced at least one vascular event, only 34% were receiving cholesterol-lowering treatment. More surprisingly, despite the fact that 78% of the study participants had elevated cholesterol, only 34% of them were receiving cholesterol-lowering treatment.
The WAFACS study found only an 18.5% reduction (mean 2.3μmol/l) in tHcy in the adherent group, where tHcy was measured (only 300 samples were assayed [5% of the total]). According to the meta-analysis by the Homocysteine Study Collaboration,7 lowering tHcy by 18.5% will reduce the risk by 8.14%, assuming a linear association. However, the reduction in tHcy in the non-adherent group is expected to be much lower than 18.5%. Thus, taking tHcy as a marker for the effect of B vitamins, the assumed sample number to detect a 20% risk reduction is far from sufficient.
Surprisingly, although the placebo group showed enhanced folate status after folate fortification (from a median of 8.8ng/ml to 15.4ng/ml), tHcy was not lowered in this group. These findings are not consistent with several studies showing approximately 10% reduction in tHcy concentrations after folate fortification.19,20 This suggests that one or more factors might have influenced tHcy in the WAFACS study. If the study failed to detect a 10% reduction in tHcy in the fortified placebo group,16,17 the ‘real’ tHcy lowering due to the treatment is only 8.5%, which reduces the expected risk lowering to 3.7% according to previous estimates from a meta-analysis.7 Furthermore, in folate-fortified populations, vitamin B12 status is a major determinant of plasma tHcy. Unfortunately, vitamin B12 status was not measured in the WAFACS study.
WENBIT was a prospective, randomized, double-blind, placebo-controlled secondary prevention study.15 It included 3,096 patients who had undergone coronary angiography. The study lasted for three years and included four groups: placebo; folic acid 0.8mg plus vitamin B12 0.4mg and vitamin B6 40mg; folic acid 0.8mg plus vitamin B12 0.4mg; or vitamin B6 40mg alone.18 This trial did not find an effect of treatment with B vitamins on total mortality or cardiovascular events.
The study included patients >18 years of age undergoing coronary angiography. Only 9.6% of the total group were above 75 years of age. In the WENBIT-90 substudy, patient age ranged from 30 to 80 years.18 Young patients have different risk profiles that are mostly related to genetic factors, and elevated tHcy is probably not a risk factor for coronary heart disease in these subjects. In a preliminary report on 90 participants in the WENBIT study,18 the authors mentioned using non-fasting blood for blood tHcy, lipids, vitamins, and other markers. In the recently published report, WENBIT included 3,096 patients treated with B vitamins for one year. However, the study did not indicate whether blood was collected under fasting or non-fasting conditions, leaving the door open for speculation about the validity of some risk factors such as lipid status and tHcy concentrations. The WENBIT study estimated that 3,088 patients were necessary to detect a 20% reduction in the primary end-point during four years of follow-up. Nevertheless, 689 participants terminated the study after less than four years; therefore, these patients cannot be included in the final analysis. As in the above-mentioned study, other confounding factors in the WENBIT study included standard medications (i.e. cholesterol-lowering) and patients with very recent events.
Statistical Power of Secondary Prevention Studies
Calculations of explanatory power show that the currently published intervention studies did not include enough subjects to provide statistically firm conclusions. Even if these studies were combined, the statistical power would not suffice to prove a reduction of cardiovascular risk. In consideration of these facts, it is no surprise that these studies did not provide positive results regarding prevention. It has been estimated that B-vitamin treatment may lead to a risk reduction of 10% in coronary heart disease. However, a meta-analysis of secondary prevention studies will not reach the required statistical power until the currently conducted intervention studies, including 50,000 subjects, are finished in several years’ time.19 However, in the case of stroke prevention the situation is different: here, the expected risk reduction is approximately 25%, thus requiring a much lower number of patients for a meta-analysis.
Significant Reduction of Stroke Risk After Recent Meta-analysis of Secondary Prevention Studies
In a recent collaborative meta-analysis of secondary prevention studies6 by American and Chinese universities, eight randomized treatment studies with B vitamins (folic acid, vitamin B6, and/or vitamin B12) were analyzed with regard to altering the risk for stroke. The metaanalysis included 16,841 patients (see Table 2). The most important result of this was a significant reduction of stroke risk (by 18%) due to treatment with folic acid. However, a reduction of stroke risk was obtained only by a treatment lasting for more than three years, the stroke risk being lowered by 29%. The reduction of stroke risk was also markedly greater than the average if the Hcy lowering was more than 20% (-23%), if the patients had no history of stroke (-25%), or if the patients consumed no folic-acid-enriched grain products (-25%). It is concluded that folic acid supplementation can effectively reduce the risk for stroke in primary and secondary prevention.
The meta-analysis by Wang et al.6 demonstrated that only intervention studies lasting longer than three years could show a significant lowering of stroke risk. The mean observation period of the HOPE-2 study was five years, while the VISP and NORVIT studies lasted for only two and three years, respectively. However, even though only relatively few strokes occurred, the HOPE-2 study could demonstrate that achieving a reduction in stroke risk took more than three years of treatment and that after five years of treatment the stroke risk was reduced by 25%.
It can be concluded that folic acid supplementation can effectively reduce the risk for stroke in primary and secondary prevention. Cardiovascular disease is a heterogeneous entity, and cardiovascular end-points can react differently to therapy with B vitamins. As the recent meta-analysis showed,6 the insufficient observation period of the studies proved to be a major restriction in terms of the explanatory power, even though earlier studies conducted on cholesterol medication showed that longer observation periods are crucial in order to obtain risk reduction.
Results from retrospective studies suggested that B-vitamin therapy might be a safe and cost-effective way to prevent stroke and cardiovascular diseases. However, the effectiveness of such therapy seems to be greater for stroke than for cardiovascular diseases.20 While the usefulness of tHcy lowering by folic acid for stroke prevention has been confirmed, no definite conclusions can be drawn concerning cardiovascular diseases until large meta-analyses of intervention studies with B vitamins are published. Current evidence suggests that the effect of tHcy lowering on CVD risk is smaller than the effect on the risk for stroke. As HHCY is particularly common in elderly patients, B-vitamin supplementation might be protective against age-related diseases. As elderly people with multiple risk factors might equally develop either CVD or stroke, physicians should realize that if vitamin B supplementation can protect against stroke, it should be recommended for all populations, regardless of the effectiveness of this treatment on CVD.