Clinical usefulness of plasma homocysteine in vascular disease

Blood specimens are usually collected in EDTA (ethylenediaminetetraacetic acid) tubes, but collection in heparinised tubes will not materially influence the results. Compared with optimally prepared plasma, tHcy levels obtained with citrated plasma are 5%–15% lower, and in serum are 5%–15% higher.4

Blood specimens should be centrifuged within 1 hour or kept on ice until centrifugation within 8 hours.4 Uncentrifuged blood that has been kept at room temperature for more than two hours is of little value.4

Chromatographic assays may be best suited for laboratories with experience in this technique, whereas enzyme assays and immunoassays are likely to be suitable for any laboratory.4

Problems with standardisation remain unresolved,4,9 particularly the lack of certified reference materials, because different types of calibrator materials often yield different results.9 Values obtained in different laboratories may therefore not be comparable.

Laboratory studies suggest that an elevated tHcy level is both atherogenic and thrombogenic.1,11-13 The proposed mechanisms by which hyperhomocysteinaemia produces complex changes in the structure and function of cerebral, coronary and peripheral vessels are listed in Box 4.

Observational studies: A recent meta-analysis by the Homocysteine Studies Collaboration of individual patient data from 12 prospective and 18 retrospective observational studies of 16 786 individuals in healthy populations found that, after adjusting for confounding caused by known vascular risk factors and correction for regression dilution caused by random variation in homocysteine measurements, a 25% higher than usual concentration of Hcy in the blood (about 3 μmol/L [0.41 mg/L]) was associated with 11% greater odds of ischaemic heart disease (odds ratio [OR], 0.89; 95% CI, 0.83–0.96), and 19% greater odds of stroke (OR, 0.81; 95% CI, 0.69–0.95).14

An independent and concurrent meta-analysis of 20 prospective studies of serum homocysteine and risk of vascular disease involving 3820 participants found that a 5 μmol/L increase in tHcy level was associated with a 32% increase in odds of ischaemic heart disease (OR, 1.32; 95% CI, 1.19–1.45) and a 59% increase in odds of stroke (OR, 1.59; 95% CI, 1.29–1.96).15

There were some inconsistencies in the results obtained by different study methods. Stronger associations between tHcy level and vascular risk were found in studies that used less robust methods, and smaller associations or no association were reported by more robust prospective cohort studies. In addition, the temporal relationship between the onset of elevated tHcy level and atherothrombotic vascular events is unclear; the finding of a stronger association in case–control studies compared with cohort studies suggests that elevated tHcy level may rise after an acute vascular event in response to tissue damage or tissue repair.16

Genetic studies: A meta-analysis of 40 observational studies involving 11 162 people with coronary heart disease and 12 758 control subjects showed that individuals with the MTHFR 677 TT genotype (MTHFR 677 C →T polymorphism of the gene that encodes the MTHFR enzyme), who have 20% higher tHcy levels than normal, have a 16% (95% CI, 5%–28%) greater risk of ischaemic heart disease than those with the CC polymorphism.17

An independent meta-analysis of 72 studies in which the prevalence of a mutation in the MTHFR gene was determined in 16 849 people showed that a 5 mmol/L increase in tHcy level was associated with a 42% increase in odds of ischaemic heart disease (OR, 1.42; 95% CI, 1.11–1.84), a 65% increase in odds of stroke (OR, 1.65; 95%CI, 0.66–4.13), and a 60% increase in odds of venous thromboembolism (OR, 1.60; 95% CI, 1.15–2.22).15

Dietary studies: Large observational studies correlating diet with long-term risk of vascular events among more than 50 000 healthy individuals suggest that a decreased dietary intake of folate is associated with an increased risk of ischaemic stroke and cardiovascular events, independent of major lifestyle and other dietary factors.18,19

Endothelial function: Folic acid has been shown to prevent postprandial endothelial dysfunction in normohomocysteinaemic subjects, and to improve endothelial function in patients with hyperhomocysteinaemia, hypercholesterolaemia, diabetes and coronary artery disease.3 The exact mechanisms underlying the ameliorative effects of folate on the endothelium are uncertain, but may include homocysteine-lowering, antioxidant actions, effects on cofactor availability, or direct interactions with the enzyme endothelial nitric oxide synthase.11-13

Carotid artery intimal thickness: Two studies have reported a decrease in the rate of progression of carotid artery intimal thickness in patients taking oral folic-acid-based multivitamins compared with before supplementation.20,21 In addition, a double-blind randomised trial of 56 stable renal transplant recipients with documented hyperhomocysteinaemia who were randomly assigned to vitamin supplementation with folic acid (5 mg daily), vitamin B6 (50 mg daily) or vitamin B12 (400 μg daily) or placebo for 6 months showed a significant decrease in carotid intima media thickness in patients who received vitamins (0.95 ± 0.20 mm [before] v 0.64 ± 0.17 mm [after]; P < 0.0001), but a significant increase in those who received placebo (0.71 ± 0.16 mm [before] v 0.87 ± 0.19 mm [after]; P < 0.05).22

Exercise electrocardiography: A double-blind, placebo-controlled, randomised trial of 158 healthy siblings of patients with premature clinical atherothrombotic disease and raised tHcy concentrations who were randomly assigned placebo or folic acid (5 mg daily) and vitamin B6 (250 mg daily) for 2 years showed that vitamin treatment was associated with a decrease in fasting tHcy level (14.7 mmol/L [placebo] v 7.4 mmol/L [vitamins]) and a decreased rate of abnormal exercise electrocardiography test results (OR, 0.40; 95% CI, 0.17–0.93; P = 0.035).23

Coronary artery restenosis and revascularisation: Following coronary angioplasty, 205 patients were allocated to placebo or the combination of folic acid (1 mg), vitamin B12 (0.4 mg), and pyridoxine (10 mg) in a randomised, double-blind trial.24 After 6 months, multivitamin treatment was associated with a significant reduction in plasma tHcy level (11.1 mmol/L [placebo] v 7.2 mmol/L [vitamins], P < 0.001) and the rate of coronary restenosis (37.6% [placebo] v 19.6% [vitamins]; P = 0.01),25 a benefit that remained evident at 1 year.26

However, the most recent study randomly allocated 626 patients undergoing coronary stenting to a single intravenous dose followed by daily oral placebo or the combination of folic acid (1.2 mg), vitamin B12 (0.06 mg), and pyridoxine (48 mg), and after 6 months patients assigned multivitamin treatment had significantly smaller minimum mean coronary artery lumen diameters, the primary end point (1.74 mm [placebo] v 1.59 mm [vitamins]), and higher rates of restenosis (27% v 35%).26

Serious clinical vascular events: The Vitamins in Stroke Prevention (VISP) Trial randomly allocated 3680 North American and Scottish patients with recent (3–120 days) non-disabling, non-cardiogenic, ischaemic stroke, who had baseline blood concentrations of homocysteine above the 25th percentile of normal, to double-blind treatment with a high-dose multivitamin formulation (including 2.5 mg folic acid, 0.4 mg cobalamin and 25 mg pyridoxine) or a low-dose formulation (including 20 μg folic acid, 6 mg cobalamin and 200 mg pyridoxine).27 After 2 years of follow-up, plasma tHcy concentration was 2 mmol/L lower in the high-dose group, but there was no significant difference in the cumulative incidence of the primary outcome event, recurrent cerebral infarction (8.8% [low dose] v 9.2% [high dose]; relative risk [RR], 1.0; 95% CI, 0.8–1.3; P = 0.68). There was also no significant reduction in any coronary event (7.4% v 7.0%; RR, 0.9; 95% CI, 0.7–1.2), death (6.9% v 5.9%; RR, 0.9; 95% CI, 0.7–1.1), and the combined outcome of ischaemic stroke, coronary heart disease or death (18.6 v 18.0; RR, 0.9; 95% CI, 0.7–1.1).27

The (unexpectedly) small between-group difference of 2 mmol/L in plasma tHcy concentration is likely to reflect the high prevalence of vitamin supplement use and widespread fortification of the grain supply and staple foods in North America with folate since the inception of the VISP trial.28 Grain and food fortification is likely to have reduced the statistical power of VISP substantially.29 Furthermore, the vitamin regimen used in VISP may have contained too little vitamin B12 in the high-dose group for elderly patients with poor B12 absorption, and too much in the low-dose group (more than the recommended daily intake), because, in the presence of folate repletion, blood concentrations of tHcy are highly dependent on vitamin B12.30 The lower than anticipated rates of recurrent strokes in both study groups and short follow-up period (only 2 years) also limited the statistical power of the VISP trial to reliably identify or exclude a modest, but clinically important, therapeutic effect of vitamins.

There are at least 12 large ongoing clinical trials involving a combined total of more than 50 000 patients which are testing the homocysteine hypothesis. These trials need to be completed, and individual patient data submitted for meta-analysis by the Homocysteine Lowering Trialists’ Collaboration (Robert Clarke, Clinical Trials Service Unit, Oxford, UK <www.ctsu.ox.ac.uk>) to more reliably and precisely estimate the effect of homocysteine-lowering treatment on the risk of atherothrombotic vascular disease, as well as its safety.

• Folic-acid-based multivitamin therapy has been proven neither effective nor ineffective in preventing clinical vascular events — there are simply not enough reliable data from large randomised controlled trials.

• Vitamin therapy is costly and potentially risky. Repeated doses exceeding 400 mg daily of vitamin B6 may cause a sensory peripheral neuropathy, and folic acid therapy may cause progressive neurological damage (subacute combined degeneration of the spinal cord) in individuals with subclinical vitamin B12 deficiency. Although the latter can be avoided by excluding B12 deficiency before starting folic acid therapy, or supplementing folic acid therapy with at least 400 mg per day of vitamin B12, it is important to not assume that folic-acid-based multivitamin therapy is safe for everyone.

• The antioxidant nutrient beta-carotene was assumed to be safe and effective in preventing atherothrombotic vascular disease until two trials in heavy smokers showed that it was associated with a higher incidence of lung cancer and higher all-cause mortality.31

• While awaiting the results and meta-analysis of the ongoing clinical trials of folic-acid-based homocysteine-lowering multivitamin therapy, insufficient evidence exists to recommend routine screening and treatment of high tHcy concentration with folic acid and other vitamins to prevent atherothrombotic vascular disease.