Homocysteine as a Risk Factor in Atherosclerotic Cardiovascular Disease

Sherif A.H. Sultan, Peadar Waters, Wael Tawfick and Niamh Hynes

Strategies focused on traditional risks factors for cardiovascular disease (CVD) such as tobacco use, hypertension, hyperglycemia, lipid abnormalities, obesity, and physical inactivity have failed to stem the tide of CVD, and investigators have begun to focus on other risk factors termed by the U.S. National Academy of Clinical Biochemistry (NACB) “emergent or new risk factors.” Based on mounting evidence from observational and epidemiologic studies, the NACB has identified homocysteine as one such emergent CVD risk factor.

The homocysteine theory of atherosclerosis first emerged in 1969, when Kilmer McCully observed that children and young adults with inborn errors of homocysteine metabolism that led to elevated levels of homocysteine in the blood developed atherosclerosis prematurely in their 2nd and 3rd decades. From this observation, investigators postulated that milder elevations of homocysteine might predispose to atherosclerotic disease. Since then, substantial clinical evidence has established that an increased risk for coronary artery disease (CAD), myocardial infarction (MI), stroke, venous thromboembolism, and peripheral vascular disease exists in patients with elevated serum levels of homocysteine.

An early meta-analysis incorporating 27 retrospective and prospective studies showed an incremental increase in risk of CAD by approximately 20% per 5 μmol/L increase in total homocysteine concentration independently of traditional CAD risk factors. They calculated an odds ratio of 1.7 for cardiovascular disease and 1.5 for cerebrovascular disease for every 5 μmol/L increase in plasma homocysteine; the odds ratio for peripheral artery occlusive disease (PAD) was 6.8. From this result, the authors extrapolated that 10% of the population’s CAD risk was attributable to hyperhomocysteinemia and that up to 50,000 deaths from CAD could be prevented annually by homocysteine level reduction. These conclusions were supported by findings from the Homocysteine Studies Collaboration meta-analysis that showed a risk reduction for ischemic heart disease by 11% and for stroke by 19% per 3 μmol/L reduction in homocysteine concentration.

These data, together with the characterization of cellular mechanisms by which homocysteine promotes oxidant stress-induced vascular dysfunction, have provided ample evidence to support clinical trials of homocysteine lowering with B vitamins as a novel therapeutic approach to patients with vascular disease.

Nomenclature

Lack of standardization and wide variation in the literature as to what constitutes abnormally elevated levels of homocysteine make direct comparison among studies difficult and might account for some of the variation in evidence for the clinical implications of hyperhomocysteinemia. It is present as protein (albumin)-bound, free circulating disulfide, and sulfhydryl forms. Current laboratory methods detect all three forms and report this as total homocysteine concentration.

In general, reference intervals published for clinical practice are not corrected for factors known to influence circulating homocysteine levels (e.g., age, ethnicity, gender) or protein-rich diets. In particular, methionine is found in high concentrations in red meat, and as the substrate for the homocysteine reactions, it directly influences homocysteine levels. In fact, a methionine-load test may be used to measure homocysteine levels in high-risk patients with normal basal levels of homocysteine to identify patients with post-load hyperhomocysteinemia. This test has been shown to uncover up to 39% of persons with homocysteine-related cardiovascular disease risk but with normal basal homocysteine levels.

Ideally, homocysteine should be measured when the patient is fasting. Many studies have simply used any value above the 95th percentile for their control group, leading to the suggested cut-off point varying from 9 to 15 μmol/L. According to the American Heart Association (AHA) advisory statement, normal fasting homocysteine concentrations range from 5 to 15 μmol/L. Intermediately elevated homocysteine levels are between 31 and 100 μmol/L, and severely elevated levels are greater than 100 μmol/L. Severely elevated levels are essentially pathognomonic for the presence of an inborn error of homocysteine metabolism, causing homocystinuria.

Pathophysiology

Homocysteine is a sulfhydryl-containing amino acid produced from the metabolism of the essential amino acid methionine. Homocysteine can undergo auto-oxidation, resulting in the formation of key biologically reactive products that participate in signaling pathways associated with increased cell toxicity. Homocysteine has been identified as a contributor to four fundamental mechanisms of disease: thrombosis, oxidant stress, apoptosis, and cellular proliferation.

There are several proposed mechanisms by which homocysteine could inflict vascular injury. Homocysteine administration has been shown to cause endothelial cell injury, in both in vitro and in vivo experimental models. Homocysteine can cyclize under acidic conditions to form homocysteine thiolactone. Hydroxyl radicals (OH⁻, OH•), and superoxide anions (O₂⁻) are byproducts of these reactions. The superoxide anion can be converted to H₂O₂ in the presence of superoxide dismutase or spontaneously undergoes dismutation to H₂O₂. Homocysteine can also promote oxidant stress by directly impairing glutathione peroxidase expression (Gpx-1), an antioxidant enzyme that reduces H₂O₂ to water. Homocysteine-induced formation of reactive oxygen species decreases levels of bioavailable nitric oxide (NO) either by reducing the availability of key NOS cofactors, such as tetrahydrobiopterin (BH₄) or by inducing conversion of NO to peroxynitrite (ONOO⁻)

By inducing oxidative stress to the endothelium, homocysteine reduces bioavailability of NO. It can also generate free radicals and inhibit the production of other antioxidants. Endothelial injury in turn results in platelet aggregation and thrombus formation. Furthermore, it impairs endothelial-mediation vasodilation and control of vascular tone. Toxic endothelial damage is also related to the stimulation of smooth muscle cell proliferation and susceptibility to oxidation of low-density lipoproteins.

Another mechanism by which homocysteine can induce vascular injury is the increased thrombogenicity mediated by increased platelet adherence and the release of platelet-derived growth factors; activated factor V, X, and XII; inhibition of protein C activation; inhibition of cell surface expression of thrombomodulin; and decreased tissue plasminogen activator activity. Homocysteine has also been thought to increase arterial stiffness by damaging elastin fibers, increasing collagen production, and stimulating smooth muscle activity

Homocysteine metabolism occurs via three pathways: remethylation of homocysteine to form methionine by methionine synthase in a vitamin B₁₂– and folate-dependent reaction; the trans-sulfuration pathway, in which, after the addition of a serine group, homocysteine is converted to cystathionine by cystathionine β-synthase (CBS), requiring vitamin B₆ as a cofactor; and in certain tissues such as liver and kidney by remethylation of homocysteine to methionine via betaine–homocysteine methyltransferase (BHMT).

Mutations in the 5,10-methyl-tetrahydrofolate reductase (MTHFR) and cystathionine β-synthase genes impair homocysteine conversion to methionine and cystathionine, respectively. In particular, a highly prevalent C677T point mutation has been associated with a thermolabile MTHFR variant. It is estimated that between 5% and 12% of the white population may be homozygous for this genotype, which results in a reduction of MTHFR activity and increased levels of plasma homocysteine. However, several studies on the clinical implications of genotype status have been inconclusive and the exact independent effects of genotype status are not yet known.

More commonly, elevated levels of homocysteine can result from nutritional deficiencies of folic acid, vitamin B₆, and vitamin B₁₂, which are key enzyme cofactors required for normal homocysteine metabolism. Mild hyperhomocysteinemia levels are seen in about 5% to 12% of the general population, and in specific populations such as alcoholic patients (as a result of poor vitamin intake), those who smoke cigarettes, or patients with chronic kidney disease this may be more common. Specifically, homocysteine levels increase markedly with age, and hyperhomocysteine rates are estimated to be as high as 30% to 40% in the elderly population.

Homocysteine and Peripheral Vascular Disease Expression

The literature to date has focused predominantly on the cardiovascular, cerebrovascular, and neurologic manifestations of hyperhomocysteinemia. Its implications in other forms of vascular disease have not been clearly elucidated. However the fact that hyperhomocysteinemia is directly implicated in endothelial dysfunction means that its adverse effects are seen right across the spectrum of vascular diseases, and there is a mounting body of evidence for its role in aortic, carotid, and peripheral disease processes.

Aortic Disease

Data available in the literature suggest a role of hyperhomocysteinemia in abdominal aortic and thoracic aortic diseases. In particular, homocysteine was investigated in patients with Marfan’s syndrome, and it was demonstrated that homocysteine levels were associated with the risk of severe cardiovascular manifestations or dissection. Homocysteine was significantly higher in patients with abdominal aortic aneurysms (AAAs) and was associated with the size of aneurysms. Some studies have found a direct association between hyperhomocysteinemia and a higher rate of AAA expansion, but the correlation is weak because of the multifactorial etiology of AAAs. Other studies have suggested that low levels of vitamin B₆, but not homocysteine, are an independent risk factor for AAA.

Studies have demonstrated that homocysteine interacts with the aortic wall by inducing both elastolysis and endothelial perturbation. In particular, pathophysiologic levels of L-homocysteine alter endothelial cell function by up-regulating expression and secretion of the proinflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in human aortic cells, suggesting that homocysteine might contribute to the initiation and progression of vascular disease by promoting leukocyte recruitment.

Cervical Artery Disease

Carotid artery intima–media thickness has been used by numerous investigators as a surrogate marker of atherosclerosis and has been shown by several to increase in patients with hyperhomocysteinemia. The clinical relevance of these thickness measures have been shown by large-scale trials, such as the Framingham study, which demonstrated a link between hyperhomocysteinemia, low folate and vitamin B₁₂ levels, and clinically significant carotid artery stenosis in the elderly. Other research has also demonstrated clinically significant carotid artery stenosis in hyperhomocysteinemic patients with concomitant symptomatic vascular disease, and separate studies have demonstrated this in patients with CAD and PAD. Additional studies have investigated higher rates of restenosis after carotid endarterectomy in patients with hyperhomocysteinemia.

Other extracranial clinical manifestations of hyperhomocysteinemia include cervical artery dissection. Cervical artery dissection, both of the carotid and vertebral arteries, is an increasingly recognized cause of ischemic stroke among otherwise healthy-appearing young and middle-aged persons. The reason these dissections occur either spontaneously or after common daily activities remains unknown, but hyperhomocysteinemia is among numerous risk factors that have been postulated (e.g., connective tissue disorders, recent infection, α₁-antitrypsin, and a variety of common neck movements). In particular, cervical artery dissection has been investigated in patients with hyperhomocysteinemia caused by polymorphisms in genes encoding for enzymes needed for homocysteine metabolism, such as CBS and MTHFR (844ins68bp CBS and C677T MTHFR genotypes) with varying strengths of association. However, a stronger relationship has been shown between mild hyperhomocysteinemia and associated low folate levels consistent with dietary deficiencies.

Reports have shown that homocysteine and inflammatory markers have a significant role in early-onset carotid atherosclerosis. Homocysteine should therefore have another effect in patients with cervical artery dissection. However, except for a few elderly cervical artery dissection patients in whom atherosclerotic lesions are described together with dissection, atherosclerotic lesions are generally not reported in younger patients who have cervical dissection and mild hyperhomocysteinemia. It seems that homocysteine produces an intimal tear with a secondary subintimal hematoma localized at a point of minor resistance without inducing wide damage. This could be a synergistic effect coupled with a minor trauma, a preexisting arterial wall defect, or both. Mild homocysteinemia seems to be responsible for CAD without producing further detectable vessel wall damage, and it is not known whether the increase recorded during cervical artery dissection and stroke is in fact a temporary condition because these patients are younger than other stroke patients, so the effects of long-term exposure to these mild homocysteine levels have not been seen.

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