Homocysteine is an amino acid produced as an intermediate

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1 CLINICAL REVIEW Homocysteine and Vascular Disease Christopher A. Friedrich, MD, PhD, and Daniel J. Rader, MD Homocysteine is an amino acid produced as an intermediate product in the metabolism of methionine, the donor of methyl groups in most methylation reactions. Extreme homocysteine elevations have been known to occur in several rare inborn errors of metabolism; these conditions are typically associated with thrombosis. Enzymes involved in this pathway use folic acid, pyridoxine (vitamin B 6 ), or cobalamin (vitamin B 12 ) as cofactors, and deficiencies of these vitamins also may affect homocysteine levels. Recently, data have been published that show an increased risk of vascular disease in patients with mild elevations of homocysteine. This paper will describe homocysteine metabolism, genetic and nutritional conditions that affect the metabolism of homocysteine, epidemiologic studies that suggest a causal link between homocysteine levels and vascular disease, and therapies. Studies show that nutritional supplementation may lower homocysteine levels, but no studies have yet been published that demonstrate any clinical outcome benefit from lowering homocysteine levels. Many publications use the term homocyst(e)ine to emphasize the sulfhydryl amino acid homocysteine as opposed to the disulfide homocystine. Mudd and Levy [1] have suggested using the term total homocysteine to signify homocysteine, homocystine, mixed disulfides that include homocysteine, and homocysteine thiolactone. In this paper we use the term homocysteine to refer to total homocysteine. Biochemistry and Metabolism Homocysteine is produced by the transfer of a methyl group from S-adenosylmethionine (SAM) to an acceptor compound, producing S-adenosylhomocysteine (SAH) [2]. Hydrolysis of this compound catalyzed by SAH hydrolase, usually an irreversible reaction, produces homocysteine that may be converted to cystathionine by the addition of serine or re-methylated back to methionine so that the cycle may continue. It is via cystathionine that cysteine may be produced. The typical reactions that produce or remove homocysteine are shown in the Figure. These pathways are usually regulated tightly, and levels of homocysteine in plasma are typically less than 10 µmol/l. The first condition found to be associated with extreme elevations of homocysteine was homocystinuria due to homozygous cystathione β-synthase deficiency. Untreated children with this condition may have free plasma homocysteine levels up to 200 µmol/l, and protein-bound homocysteine may be elevated as well. The primary vascular manifestation of homocystinuria is thromboembolism, which can occur in any vessel, arterial or venous [3,4]. Genetic Causes of Hyperhomocysteinemia Although nutritional deficiencies are felt to be the most common causes of mild elevations of homocysteine levels, the most striking elevations are seen in some rare inborn errors of metabolism. Studies of patients with these conditions led to the initial observations of the association of hyperhomocysteinemia and vascular disease. In a study of 629 patients with homocystinuria, Mudd et al [5] classified patients as to whether or not treatment with vitamin B 6 (pyridoxine) lowered homocysteine levels. Among untreated B 6 -responsive and B 6 -nonresponsive patients, the chance of having a clinically detected thromboembolic event by age 15 years was 12% and 27%, respectively. Mudd et al also found that treatment of B 6 -responsive patients who were detected late reduced the incidence of initial thromboembolic events. Curiously, of those patients who underwent a total of 586 surgical procedures, only 25 postoperative thromboembolic events occurred, 6 of them fatal. Regarding the risk of vascular events of those heterozygous for cystathionine β-synthase deficiencies, Mudd et al [6] reported on 203 families and found no evidence of increased myocardial infarctions or cerebrovascular accidents in parents or grandparents of probands. A subsequent study indicated that heterozygotes have serum homocysteine levels about 3 times the population average and have a higher risk for cardiovascular disease [7]. More recently, it has been reported that thrombosis in patients with cystathionine β-synthase deficiency is increased if they also are affected by the factor V Leiden mutation, which affects factor C resistance [8]. Of 7 unrelated families with Christopher A. Friedrich, MD, PhD, Assistant Professor of Medicine, Department of Medicine, Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA; and Daniel J. Rader, MD, Director, Preventive Cardiology, Department of Medicine, Division of Cardiovascular Medicine and Experimental Therapeutics, University of Pennsylvania School of Medicine. Vol. 6, No. 6 JCOM June

2 HOMOCYSTEINE AND VASCULAR DISEASE Protein Glycine Serine Tetrahydrofolate Methionine N,N-Dimethylglycine S-Adenosylmethionine CO 2 Acceptor [Decarboxylated S-adenosylmethionine] S-Adenosylhomocysteine 5,10-Methylenetetrahydrofolate 5-Methyltetrahydrofolate Betaine Choline Homocysteine Serine Cystathionine Methylated acceptor α-ketobutyrate Cysteine SO 4 Figure. Metabolic pathways that involve homocysteine. homocystinuria, thrombosis occurred in 6 of 11 patients with homocystinuria between the ages of 0.2 and 8 years. All 6 patients also had the factor V Leiden mutation. Of 4 additional homocystinuria patients aged 1 to 17 years who did not have the factor V Leiden mutation, none had thrombosis. Other genetic causes of elevated homocysteine levels include (1) vitamin B 12 malabsorption (Imerslund syndrome), (2) transcobalamin II deficiency, (3) vitamin B 12 metabolic defect, (4) N(5,10)-ethylenetetrahydrofolate reductase deficiency, (5) methylcobalamin deficiency, cbl G type, and (6) vitamin B 12 -responsive homocystinuria, cbl G type (Table). Nutritional deficiencies of folate or vitamin B 12 may cause elevations of homocysteine, as can impaired renal excretion of homocysteine. Iatrogenic causes of hyperhomocysteinemia include administration of methotrexate, 6-azauridine triacetate, or isonicotinic acid hydrazide. Of importance in patients at risk for vascular disease is the observation that administration of colestipol with niacin led to a mild elevation of homocysteine levels in treated patients as compared to placebo-treated control subjects (15.8 µmol/l versus 10.6 µmol/l) [9]. A relatively common genetic disorder is a thermolabile variant of 5,10-ethylenetetrahydrofolate reductase. About 10% of the population is homozygous for this variant, which leads to moderate increases in plasma homocysteine concentration [10 13]. Studies of the clinical significance of this variant have been inconclusive. In those who were determined to be homozygous based on their phenotype (ie, thermolabile 5,10-methylenetetrahydrofolate reductase), the combined odds ratio was reported as 3.33 (95% CI, 2.01 to 5.53) [14]. However, in studies in which cases were determined by genotype (ie, the C677T mutation responsible for the phenotype), no increased risk was found [14]. Other studies have shown that for those with the mutation, homocysteine levels are typically higher if blood folate levels are also low [11,15]. A recent study of 3524 children enrolled in the Child and Adolescent Trial for Cardiovascular Health measured nonfasting serum total homocysteine levels. The range of levels was 0.1 to 25.7 µmol/l (median, 4.9 µmol/l), with geometric mean levels greater in boys than girls, greater in blacks than whites or Hispanics, greater in nonusers of multivitamins 38 JCOM June 1999 Vol. 6, No. 6

3 CLINICAL REVIEW than users, and greater in smokers than nonsmokers. Serum homocysteine levels were inversely correlated with levels of folic acid, vitamin B 12, and vitamin B 6. There was no association with serum lipid levels or with a family history of cardiovascular disease. Osganian et al [16] concluded that although homocysteine levels are much lower in children than in adults, a small percentage of children may be predicted to be at increased risk for cardiovascular disease as adults. They found 5% of their subjects had homocysteine levels greater than 9 µmol/l, the level at which Nygard et al [17] found the risk of death began to increase in patients with coronary artery disease. Table. Genetic Conditions Associated with Elevated Homocysteine Levels Condition McKusick Number Cystathionine β-synthase deficiency N(5,10)-methylenetetrahyrofolate reductase deficiency Intestinal malabsorption of vitamin B Transcobalamin II deficiency Vitamin B 12 metabolic defect, type I Vitamin B 12 metabolic defect, type II Vitamin B 12 -responsive homocystinuria, cbl E type Methylcobalamin deficiency, cbl G type Vascular Effects of Mild Hyperhomocysteinemia A meta-analysis of 27 studies showed the odds ratio for coronary artery disease risk was increased by 1.6 (95% CI, 1.4 to 1.7) in men and by 1.8 (95% CI, 1.4 to 2.3) in women for each 5-µmol/L increase in homocysteine [18]. In the Multiple Risk Factor Intervention Trial, no association was found between homocysteine concentrations and ischemic heart disease [19]. Although 1 prospective study [20] failed to show an association between homocysteine levels and vascular disease risk, most other prospective studies have confirmed a strong, independent association between vascular disease risk and elevations of homocysteine [21 26]. Nygard et al [17] studied patients with coronary artery disease and found the adjusted odds ratio for risk of death among those with homocysteine levels from 9.0 to 14.9 µmol/l was 1.9 when compared to patients with homocysteine levels below 9 µmol/l; for those with levels from 15.0 to 19.9 µmol/l, the adjusted odds ratio for death was 2.8. This is the only large study of a population with established coronary artery disease. A large multicenter European study assessed the vascular disease risk attributable to homocysteine. In this casecontrol study, 750 patients with cardiac, cerebral, or peripheral vascular disease were compared to 800 control subjects. All participants were younger than age 60 years, and both sexes were included. Homocysteine was measured in the fasting state and after a methionine load, and the geometric mean homocysteine levels were significantly higher in cases than controls under both conditions. Those whose plasma homocysteine levels were in the top quintile of the control distribution (ie, homocysteine level at least 12 µmol/l) were compared to the remaining subjects, and their relative risk for vascular disease was 2.2 (95% CI, 1.6 to 2.9) [27]. Methionine loading identified an additional 27% of at-risk patients when the top quintile was defined as those subjects whose post-methionine levels were at least 38 µmol/l. They noted interaction effects with hypertension and smoking in both sexes. Erythrocyte folate and serum cobalamin and pyridoxine levels were inversely correlated with homocysteine levels, although only serum pyridoxine levels were significantly lower in cases than in controls. Although the number of users of vitamin supplements was small, these patients seemed to have a markedly lower risk of vascular disease, partially attributed to lower homocysteine levels. Their relative risk was 0.38 (95% CI, 0.2 to 0.72). The authors concluded that both elevated fasting and post methionine load homocysteine levels were as strongly related to vascular disease as serum cholesterol level and cigarette smoking. Wald et al [14] conducted a prospective study of 21,520 British men aged 35 to 64 years. In a nested case-control study, they analyzed results from 229 men without a prior history of ischemic heart disease on entry into the study who subsequently died of ischemic heart disease and compared these results to those from 1126 age-matched control subjects. The authors determined serum homocysteine levels rose 0.75 µmol per decade (95% CI, 0.45 to 1.06 µmol). They reported that serum homocysteine levels were significantly higher in those who died from ischemic heart disease (13.1 versus 11.8 µmol/l). They compared men in the highest quartile of serum homocysteine levels to those in the lowest quartile and found the risk for ischemic heart disease was 3.7 times higher (95% CI, 1.8 to 4.7). After adjusting for other risk factors, the risk was 2.9 times higher. They found a continuous dose-response relationship and estimated there was a 41% increase (95% CI, 20% to 65%) in risk for each 5 µmol/l increase in serum homocysteine concentration. The increase was 33% (95% CI, 22% to 59%) after adjustment for blood pressure and apolipoprotein B levels. Ridker et al [28] studied 28,263 participants in the Women s Health Study and determined 122 women who developed cardiovascular events while in the study and compared them to 244 age- and smoking-matched controls. Subjects with homocysteine levels in the highest quartile had Vol. 6, No. 6 JCOM June

4 HOMOCYSTEINE AND VASCULAR DISEASE a 2-fold increase in risk of any cardiovascular event (relative risk, 2.0; 95% CI, 1.1 to 3.8). For subjects with homocysteine levels above the 95th percentile (20.7 µmol/l), the relative risk was 2.6 (95% CI, 1.1 to 5.7). Risk estimates were independent of traditional risk factors and greatest for the endpoints of stroke and myocardial infarction. They also found self-reported multivitamin supplementation was associated with reduced homocysteine levels (P < 0.001). In contrast to these studies are the findings reported by Folsom et al [29] in the Atherosclerosis Risk in Communities (ARIC) Study. In a prospective case-cohort study of 15,792 persons aged 45 to 64 years from 4 U.S. communities, investigators found no asssociation between elevated homocysteine levels and cardiovascular risk after adjustment for other risk factors. They did find an association between plasma pyridoxinal 5'-phosphate levels and the incidence of coronary heart disease over an average follow-up period of 3.3 years. In genetic studies, they also found no association between coronary heart disease and the C677T genotype for the 5,10-methylenetetrahydrofolate reductase gene, nor with 3 mutations of the cystathionine β-synthase gene. Screening and Diagnosis A position paper on the use of total plasma homocysteine measurements has been published by the American Society of Human Genetics and the American College of Medical Genetics [30]. The authors note that most of the homocysteine in plasma is bound to albumin and other proteins, and the techniques used to measure levels of free amino acids may be inadequate to detect small increases in free homocysteine levels. They note that plasma must be placed on ice immediately to prevent leakage of free homocysteine from erythrocytes, and the sample centrifuged and the plasma frozen in less than 30 minutes after collection. The total homocysteine is then stable for months. They also recommend the addition of a reducing agent (eg, dithiothreitol or β-mercaptoethanol) to fresh plasma to release homocysteine from plasma proteins before measurement. Because of the many complexities of sample handling and homocysteine measurement, the authors recommend any single elevated homocysteine value be evaluated by repeating the collection after at least an 8-hour fast. Their recommendation is that the underlying etiology of an elevated homocysteine level always be determined before any treatment is initiated. They recommend measurements of serum levels of folate and cobalamin, erythrocyte levels of folate, and serum and/or urine methylmalonic acid levels. They emphasize that supplementation with folic acid may prevent the identification of pernicious anemia by masking the typical hematologic findings while allowing the neurologic problems to progress. They do not provide any cost/ benefit analysis of this model compared with an alternative (eg, empiric supplementation of folate for those with elevated homocysteine levels), reserving more intensive evaluations for those who fail to respond to treatment. Although many patients may be found to have an elevated homocysteine level on a random fasting plasma sample, additional hyperhomocysteinemic subjects may be found after a methionine challenge. In the latter test, a patient is given oral methionine (100 mg/kg), and blood is collected 6 hours later. Challenging the metabolic pathway with a large bolus of the precursor to homocysteine may reveal those who may have intermittent elevations of homocysteine but clearly poses a logistical problem for routine screening studies. Bostom et al [31] showed that using fasting homocysteine levels alone would have led to the failure to identify about 40% of their subjects, and Graham et al [27] reported that had they not used methionine loading, they would have failed to identify 27% of their subjects. No data exist that show a consistent association between post methionine load homocysteine levels and cardiovascular risk, and the logistic difficulties involved in pre and post methionine loading measurements make this unlikely to be useful as a screening tool. Treatment Most trials have centered on vitamin supplementation, especially using folate. By increasing the availability of vitamin cofactors, the enzymes that catalyze the recycling of homocysteine back to methionine may be stimulated, lowering homocysteine levels. Homocysteine levels have been found to decrease by as much as 40% after dietary supplementation with folic acid [16,32,33]. However, Boushey et al [18] reviewed 9 intervention trials and found a plateau of homocysteine-lowering at a dose of 400 mg per day, with little additional change in homocysteine levels at higher doses of folic acid. Ward et al [34] reported supplementation in middle-aged patients with this dose of folic acid resulted in decreases of homocysteine of 1.9 µmol/l. Several other studies have confirmed the effect of folic acid on homocysteine levels [35 37]. The U.S. Food and Drug Administration, in an effort to reduce the incidence of neural tube defects such as spina bifida, has mandated that grain cereal products be fortified with folic acid, with 140 mg of folic acid per 100 g of cereal or grain product. Malinow et al [38] measured the effect of foods fortified with 499 mg or 665 mg of folic acid per 30 g of cereal in a randomized, placebo-controlled, double-blind crossover trial. They studied 75 men and women with coronary artery disease who were taking the recommended dietary allowances of vitamins B 6 and B 12 and found that fortification with folic acid at levels greater than currently recommended by the FDA may be useful to prevent vascular disease. Jacques et al [39] recently showed fortification 40 JCOM June 1999 Vol. 6, No. 6

5 CLINICAL REVIEW was associated with an improvement in folate status in middle-aged and older adults. It is difficult to reconcile the observations of Malinow et al with those of Jacques et al. In the former study they found fortification at the FDArecommended level was not sufficient to lower homocysteine levels in men and women with established coronary artery disease. In the latter study, fortification was observed to lower homocysteine levels in the Framingham cohort. Treatment with pyridoxine has been helpful in some patients with homocystinuria, and treatment with cobalamin may lower homocysteine levels caused by vitamin B 12 deficiency. Treatment with betaine (trimethylglycine) has been demonstrated to lower homocysteine levels in homocystinuria but has not been studied in subjects with mild elevations of homocysteine [40,41]. Betaine provides methyl groups that are added to homocysteine by betainehomocysteine methyltransferase to produce methionine. The American Heart Association Nutrition Committee has recently published a review of homocysteine [42]. They note the lack of evidence that reduction of plasma homocysteine is associated with a reduction in cardiovascular disease. They recommend increased consumption of dietary folate through foods such as vegetables, fruits, legumes, meats, fish, and fortified grains and cereals. They do not recommend general population screening for hyperhomocysteinemia, but for patients with traditional risk factors for cardiovascular disease they suggest measuring fasting homocysteine levels. Other patients at risk for elevated homocysteine levels are those with malnutrition (including malabsorption syndromes), systemic lupus erythematosus, or renal failure, all of which can affect folate absorption. Similarly, treatment with L-dopa, bile-acid sequestrants, nicotinic acid, theophylline, and methotrexate can reduce folate levels. Future Directions A substantial body of evidence seems to implicate elevated levels of homocysteine as a causative factor for vascular disease. Although initial studies appeared to demonstrate a threshold effect in which homocysteine levels above a certain value were associated with increased cardiovascular risk, further studies have shown the risk seems to be increased in a dose-response manner with the risk gradually increasing when levels exceed 9 µmol/l. Intervention studies have shown it is possible to lower homocysteine levels by vitamin supplementation, but no studies have yet been conducted to determine if lowering homocysteine levels will decrease clinical events. Nor have studies addressed other unusual clinical relationships, such as the effect of factor V Leiden heterozygosity on mild hyperhomocysteinemia. Treatment with vitamin supplements is widely available, inexpensive, and free of side effects. 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