VIEWS AND REVIEWS IN VASCULAR MEDICINE AND BIOLOGY

Homocysteinemia as a Risk Factor for Atherosclerosis: A Review Mark R. Nehler, MD, Lloyd M. Taylor, Jr, MD, and John M. Porter, MD Department of Surgery, Division of Vascular Surgery, Oregon Health Sciences University, Portland, Oregon

11 Elevation in plasma homocysteine has been widely studied as an independent risk factor for atherosclerosis. Animal laboratory models have demonstrated rapid onset vascular lesions with homocysteine infusion. A large body of data indicates a consistent relationship between plasma homocysteine and symptomatic atherosclerotic disease involving the coronary, peripheral, and cerebral circulations. Elevated plasma homocysteine can be predictably normalized with oral folate in most patients. Despite the wealth of published clinical data on this topic, it is unknown if normalization of plasma homocysteine in patients with symptomatic atherosclerosis will prevent or arrest the disease process. © 1997 by Elsevier Science Inc. Cardiovasc Pathol 1997;6:1–9

Complications of atherosclerosis continue to be the leading cause of death and disability in industrialized nations. Despite a widespread research effort spanning decades, there remains no clearly defined cause or cure for this disease that directly or indirectly affects the lives of almost all individuals in the western world. Autopsy findings consistently show atherosclerosis is present to some degree in nearly all aged people, suggesting it should be regarded both as a normal process of aging as well as a distinct disease entity. Investigation of atherosclerosis etiology requires a distinction between atherosclerosis as a normal consequence of aging and atherosclerosis as a disease entity causing disability and/or death. Atherosclerosis is most appropriately regarded as a disease when associated with both rapid progression and clinical symptoms. Widely accepted risk factors for atherosclerotic disease include advanced age, diabetes, tobacco use, arterial hypertension, hypercholesterolemia, hypertriglyceridemia, decreased high-density lipoprotein, hypercoagulability, sedentary lifestyle, and elevated plasma homocysteine. Clearly the study of lipid metabolism has dominated research into atherosclerosis etiology for decades, although now it is widely recognized that a

Manuscript received April 16, 1996; accepted June 3, 1996. Address for reprints: Dr. Lloyd M. Taylor, Jr., Department of Surgery, Division of Vascular Surgery (OP-11), Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. Cardiovascular Pathology Vol. 6, No. 1, January/February 1997:1–9  1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

large number of people with symptomatic atherosclerotic disease have no detectable evidence of abnormal lipid metabolism (1,2). In the last 10 years, elevation in plasma homocysteine has been widely studied as an independent risk factor for atherosclerosis. A description of the metabolism of homocysteine, its relationship to vascular disease, evidence supporting its role as an independent risk factor for atherosclerotic vascular disease, and potential roles of treatment will form the basis for this review.

Homocysteine Metabolism Homocysteine is a sulfur-containing amino acid not included in the 20 essential amino acids that serve as structural elements of all proteins. It exists in three forms in human plasma: as homocysteine, as the disulfide homocystine, and as the mixed disulfide homocysteine-cysteine (Figure 1). Almost all plasma homocysteine is bound to proteins. The functional metabolic role of homocysteine is as an intermediary after the demethylation of dietary methionine during the formation of cysteine or during remethylation to form methionine (Figure 2). In the former, the enzyme cystathionine b-synthase with pyridoxine as a vitamin cofactor catalyzes the reaction of homocysteine with serine to form cystathionine, which is then cleaved to form cysteine. This series of reactions resulting in the conversion of the four-carbon dietary amino acid methionine to the three-carbon amino acid cysteine is called the trans-

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Figure 1. Forms of homocysteine found in human plasma: homocysteine, homocysteine-cysteine mixed disulfide, and homocystine. (Used with permission of publisher, from Masser PA, Taylor LM Jr, Porter JM. Importance of elevated plasma homocysteine as a risk factor for atherosclerosis. Ann Thorac Surg 1994; 58:1240–1246.)

sulfuration pathway. A deficiency of the enzyme cystathionine b-synthase results in abnormal accumulation of homocysteine. Homocysteine may also be remethylated to form methionine via the enzymes methyltransferase and methylenetetrahydrofolate reductase. This step requires a methyl group from cofactors folate, cobalamin, betaine, or choline. Deficiencies in any of these enzymes or co-factors may result in an abnormal elevation of plasma homocysteine.

These pathways are collectively referred to as the remethylation pathway. It has been postulated that the concentration of the intermediary S-adenosyl methionine acts to regulate the intracellular metabolism of homocysteine, including both degradation to cysteine or remethylation to methionine through inhibition of methylene-tetrahydrofolate reductase and stimulation of cystathionine b-synthase (3). A defect in this reg-

Figure 2. Homocysteine metabolism in man. Enzymatic reactions that are regulated by S-adeonsylmethionine (SAM) are indicated by large arrows; closed arrows indicate inhibition, open arrow indicates activation. Enzymes: (1) homocysteine methyltransferase, (2) methylene tetrahydrofolate reductase, (3) betaine methyltransferase, (4) choline dehydrogenase, (5) cystathionine b-synthase, (6) g-cystathionase. THF 5 tetrahydrofolate, PLP 5 pyridoxal-59-phosphate. (Used with permission of publisher, from Selhub J, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation of S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 1992;55:131–138.)

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ulation has been postulated to be responsible for the elevation in homocysteine observed when only one enzymatic limb of homocysteine metabolism is deficient.

Homocysteine and Vascular Disease The inborn error of metabolism, homocystinuria, was first described in 1962 (4,5). In this rare disorder, homocysteine accumulates in the plasma and tissue, spilling into the urine in large quantities. The patients have multiple abnormalities, including dislocated lens, mental retardation, skeletal disorders, and severe premature vascular disease. The vascular manifestations include virulent atherosclerotic plaque formation in addition to widespread arterial and venous thrombosis usually resulting in death as early as the first decade (6). Whereas a homozygous defect in the enzyme cystathionine b-synthase is the most frequent etiology (7,8), homocystinuria variants with minimal levels of methyltransferase (9) and methylene-tetrahydrofolate reductase (10) with similar vascular manifestations have been described. Similar vascular pathology observed in patients with elevated plasma homocysteine levels secondary to disparate enzymatic defects suggests that homocysteine is the causative agent. Animal models using homocysteine infusions have duplicated the rapid onset vascular lesions (11). Treatment with pyridoxine, folic acid, or both in homocysteinemic patients decreases serum homocysteine (11–13). Clearly the potential for effective treatment of this risk factor for atherosclerosis motivates the current research fervor.

Clinical Data Early studies of plasma from patients with homocystinuria revealed multiple abnormal sulfur-bearing amino acids, including homocysteine-cysteine mixed disulfide which

at that time were undetectable in normal plasma (14). Only later were these metabolites found in normal plasma at very low concentrations (15–17). Due to technical problems with detection sensitivity, multiple clinical evaluations to determine the relationship between plasma homocysteine and symptomatic atherosclerosis have measured homocysteine levels in response to an oral methionine load (18,19). An abnormal response (defined as $90th percentile of normal plasma homocysteine) identifies patients with significant homocysteinemia. Presently, measurement of the total plasma homocysteine level using high performance liquid chromatography (HPLC) permits detection of abnormal levels without the need for methionine loading, thus significantly simplifying patient screening evaluation. We have observed that patients demonstrate an increase in plasma homocysteine with methionine loading, which is proportional to their baseline level. The reproducibility of plasma homocysteine regardless of normal dietary intake indicates that fasting is not necessary. (20). This finding has been reproduced by others (21). At present, there is no universally accepted “standard” assay technique for plasma homocysteine. Many investigators use HPLC, but with multiple technical differences (22–32). Accordingly, no accepted range for normal plasma homocysteine has been defined. There is general interlaboratory agreement that men have higher values than women, and homocysteine levels in postmenopausal women are higher than those in premenopausal women (30,33,34). The ranges for normal values determined in multiple investigations are listed in Table 1. To date, normal values have been based on control populations defined by their asymptomatic status, without more detailed noninvasive studies to exclude occult atherosclerotic lesions. Another confounding factor is the variety of conditions demonstrated to potentially affect plasma homocysteine levels. Studies of fraternal and identical twins suggest that

Table 1. Normal Plasma Homocysteine Values Reference 22 22 22 22 30 30 34 34 55 60 60 59 46 32 32

Subjects (n)

Mean Homocysteine (SD)

Asymptomatic men ,60 yrs (35) Asymptomatic men ,60 yrs (18) Asymptomatic women ,60 yrs (39) Asymptomatic women ,60 yrs (11) Asymptomatic men, mean age 34 (36) Asymptomatic women, mean age 34 (35) Asymptomatic men, mean age 61 (34) Asymptomatic women, mean age 61 (32) Male controls (36) Asymptomatic controls (both sexes) (45) Hypertensive controls (45) Framingham asymptomatic males (255) Asymptomatic (both sexes, mean age 61) (31) Males, mean age 39 (12) Females, mean age 37 (12)

11.2 (3.6) 10.7 (2.1) 8.6 (2.8) 9.0 (2.2) 9.3 (1.9) 7.9 (2.3) 12.7 (2.5) 11.1 (3.6) 13.5 (3.6) 7.3 (2.9) 9.9 (4.1) 10.9 (4.9) 10.7 (3.2) 15.8 (6.4) 16.5 (4.4)

All values given as micromoles/liter homocysteine.

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homocysteine levels may be genetically controlled (35). Deficiencies in the co-factors folate (30,36), pyridoxine (37), and vitamin B12 (30,38,39) have been demonstrated to elevate plasma homocysteine (40). Analysis of vitamin levels in older patients from the Framingham study indicates that the majority of patients with elevated plasma homocysteine in this group may suffer relative vitamin deficiencies (41). Plasma homocysteine is inversely related to renal function, with several studies demonstrating elevated levels in patients undergoing hemodialysis (42–45). Elevated plasma homocysteine has also been reported in association with elevated uric acid and/or diuretic use (21,46,47), probably reflecting the influence of renal function. This relationship may prove especially important, as traditional cardiovascular risk factors have not adequately explained the excess burden of cardiovascular disease observed in the population with end-stage renal disease (48). Evidence exists for hormonal regulation of plasma homocysteine in women. As noted above, postmenopausal women have higher plasma homocysteine levels than premenopausal women do. A single report has demonstrated reduction in plasma homocysteine in postmenopausal women using hormonal replacement therapy (49). In addition, diminished homocysteine levels have been documented in pregnancy (50,51). Although most studies in patients with symptomatic atherosclerosis show no relationship between plasma homocysteine and hypertension, two investigations in patients without symptomatic atherosclerosis demonstrated correlation of plasma homocysteine with elevated systolic and diastolic blood pressure (52,53). A large body of data indicates a consistent relationship between plasma homocysteine and symptomatic atherosclerotic disease involving the coronary, peripheral, and cerebral circulations. (33,34,46,47,54–61). The results of a number of screening studies are shown in Table 2. Mean plasma homocysteine values have been 25% to 50% higher in patients with symptomatic atherosclerotic disease compared to controls. In addition, most series demonstrate a subset includ-

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ing 15% to 40% of the symptomatic patients with markedly elevated plasma homocysteine ($90–95th percentile). All studies to date have demonstrated elevations in plasma homocysteine occur independently of other recognized risk factors for atherosclerotic disease. A few studies have sought a correlation between abnormally high plasma homocysteine values in patients with symptomatic atherosclerosis and corresponding abnormalities in the known co-factors of homocysteine metabolism: folate and vitamins B6 and B12. Molgaard et al. (58) found that elevated plasma homocysteine levels occurred almost exclusively in patients with folate levels ,11 nmol/l. Brattstrom et al. (38) demonstrated that 40% of the variability in plasma homocysteine could be accounted for by age, cofactor levels, and abnormal renal function using logistic regression analysis. Lewis et al. (62) demonstrated that folate levels higher than “normal” values were necessary to prevent elevated plasma homocysteine in patients with coronary artery disease. More recently, Selhub et al. (63) examined homocysteine and vitamin levels in 1,160 patients from the Framingham study. Thirty percent of these patients had homocysteine levels above the 90th percentile, and twothirds of these could be explained by relative vitamin deficiency. Work is ongoing to define the potential genetic contribution to homocysteinemia, including the development of genetic markers to determine patients at risk. The prevalence of moderate hyperhomocysteinemia in the general population is estimated at 5% to 7% (64,65). Homozygous or heterozygous conditions for the thermolabile defect in methylenetetrahydrofolate reductase (66) or heterozygous cystathionine b-synthase deficiencies are the most frequently observed genetic causes. These genetic defects cause about 50% reduction in corresponding enzyme activity and are estimated to occur at a prevalence of about 5% in the general population (67). However, not all individuals with these deficiencies demonstrate elevated plasma homocysteine, suggesting other necessary conditions for full phenotypic expression

Table 2. Studies Confirming Elevated Plasma Homocysteine as a Risk Factor for Arterial Disease Year

Reference

Patients (n)

Patient Mean

Control Mean

% with Elevated HC

1985 1988 1989 1990 1990 1991 1991 1992 1992 1993 1994

56 54 60 57 46 59 47 58 34 33 61

Peripheral artery disease (47) Myocardial infarct (21) Stroke (45) Coronary Disease (64) Stroke (41) Coronary disease (176) Cerebral 1 peripheral (214) Claudication (78) Stroke (142) Diabetic microangiopathy (52) Coronary artery disease (266)

16.1 16.4 13.1 13.1 15.8 13.9 14.3 16.7 18.6 10.8 12.0

10.1 13.5 7.3 11.3 10.7 10.9 10.1 13.8 11.9 7.5 10.1

47 24 N/G 19 N/G 28 39 23 40 N/G 17.6

All values given as micromoles/liter of homocysteine. N/G 5 not given.

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(68,69). Genest et al. (59) evaluated plasma homocysteine in family members of patients with homocysteinemia and symptomatic coronary disease. Half of these patients had first degree relatives with elevated plasma homocysteine. A genetic defect responsible for methylene-tetrahydrofolate reductase deficiency has been isolated and proposed to represent a genetic risk factor for vascular disease (70). Due to the natural history of the homozygous state, possible relationships with familial patterns of coronary disease, and the implications regarding dialysis patients, speculation has focused on elevated plasma homocysteine as a marker for a particular virulent form of atherosclerosis. To date, only a retrospective study at Oregon Health Sciences University in 214 patients with symptomatic lower extremity or cerebral vascular disease has addressed this issue (47). Patients with elevated plasma homocysteine were significantly more likely to have clinical progression of peripheral and coronary arterial disease or vascular laboratory determined progression of their lower extremity arterial disease than were patients with normal plasma homocysteine (Figure 3). In addition, the rate of clinical progression of disease was more rapid in patients with elevated plasma homocysteine (Figure 4). Multiple regression analysis indicated that homocysteine correlated with progression independent of other established atherosclerotic risk factors. In a case-control study, elevated plasma homocysteine levels were demonstrated to increase the likelihood of carotid artery intimalmedial wall thickness in asymptomatic adults (71). A recent study examined homocysteine levels in blood samples obtained prospectively during the ongoing Physi-

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cians Health Study. This trial demonstrated a relative risk of 3.1 for myocardial infarction in men with homocysteine values at or above the 90th percentile (72). This is the first study to identify elevated plasma homocysteine as a significant risk factor for atherosclerosis in a prospective manner (although analyzed retrospectively). We are currently involved in a prospective study of plasma homocysteine and the progression of peripheral and cerebral vascular disease (73).

Basic Science Data Homocysteine produces atherosclerotic vascular lesions when infused in experimental animals (11). A number of studies have investigated potential mechanisms for the atherogenicity of homocysteine. These studies have primarily focused on the potential production of toxic byproducts by the thiol side group of the homocysteine molecule. Multiple investigations of human umbilical vein endothelium and human venous endothelium cultures have detected direct and indirect evidence of structural and functional alterations in response to elevated homocysteine concentrations. McCully has not only demonstrated a direct toxic effect of homocysteine on cultured endothelial cells (74) but has shown that elevated plasma homocysteine acts in concert with dietary lipids (75). He has suggested homocysteine accumulation results in abnormal sulfuration of proteoglycans through the intermediary homocysteine thiolactone, a known cellular toxin (76,77). Using cultured endothelial cells from normal subjects, Wang et al. (78) noted measurable activity for only two of

Figure 3. Presence of clinical and laboratory evidence of progression of disease in patients with normal (hatched bars) and elevated (solid bars) plasma homocysteine. Clin 5 clinical progression, LED 5 lower extremity disease, CVD 5 carotid artery disease, CAD 5 coronary artery disease. Percent refers to percentage of all patients in each category showing evidence of disease progression. (Used with permission of publisher, from Taylor LM Jr, Porter JM. Elevated plasma homocysteine as a risk factor for atherosclerosis. Chapter in Porter JM, guest ed. Nonatherosclerotic Disease. In: Rutherford R, ed. Seminars in Vascular Surgery. Philadelphia: WB Saunders, 1993;6:36–45.)

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Figure 4. Rate of clinical progression of disease (lower extremity plus carotid artery plus coronary artery disease) in patients with elevated plasma homocysteine (solid bar) compared to patients with normal plasma homocysteine (hatched bar), p , .002. CPI 5 clinical progression index (calculated by dividing the length of clinical follow-up in months by the number of clinical progression events. (Used with permission of publisher from Taylor LM Jr, Porter JM. Elevated plasma homocysteine as a risk factor for atherosclerosis. Chapter in Porter JM, guest ed. Nonatherosclerotic Disease. In: Rutherford R, ed. Seminars in Vascular Surgery. Philadelphia: WB Saunders 1993;6:36–45.)

the three cellular enzymes involved in intracellular homocysteine metabolism. Activity level for methyltransferase was negligible. Due to this inherent restriction of the remethylation pathway in endothelial cells, he postulated they would be less able to process elevated plasma homocysteine if the transsulfuration pathway was impaired, as in the heterozygous state for cystathionine b-synthase deficiency. This would lead to elevation in intracellular homocysteine and resultant cellular injury. In another study, cultured bovine endothelium monolayers became permeable to labeled albumin when homocysteine and copper were added to the medium, indicating oxidative damage to the endothelium (79). This process was inhibited by catalase, suggesting hydrogen peroxide was the oxidant agent produced. Further studies using this model demonstrated endothelial cell lysis with homocysteine and copper exposure over time, again inhibited by catalase (80). Despite this, in vivo studies have not demonstrated any difference in systemic peroxidation byproducts in small numbers of patients with homocysteinemia compared with controls (81,82). The activity of several endothelial cell surface anticoagulants appears impaired by elevated homocysteine. In vitro models of homocysteinemia have demonstrated a reduction in the measured activity of protein C (83), possibly due to reduced activity of thrombomodulin (cofactor in protein C activation) (84). Alteration in endothelial cell surface proteins may be due to defects in intracellular transport secondary to oxidative changes caused by homocysteine (85). Ex-

cess homocysteine has been demonstrated in vitro to increase platelet-derived thromboxane B2 and reduce endothelial-derived prostacyclin (86). Early structural changes in the endothelium resembling those observed in atherosclerosis have been documented. Using a pig model, homocysteinemia produces loss of elastic lamina and hypertrophy of muscle cells in the vessel media. These effects were partially eliminated with the use of a captopril/thiazide regimen (87). In summary, these studies suggest that homocysteinemia produces endothelial cell injury and may induce a thrombotic state. Additional information is awaited.

Prevention and Treatment Current recommendations regarding atherosclerosis prevention include modification of lipid status through diet and/or medical therapy, cessation of smoking, hypertension control, and moderate exercise programs. These require major changes in lifestyles and addictive behavior patterns, thus severely limiting overall success in large populations. Abundant information indicates elevated plasma homocysteine can be predictably normalized with oral folate in most patients (88–90). Those with homocysteine levels resistent to oral folate generally respond to nontoxic doses of pyridoxine, vitamin B12, choline, or betaine (91,92). Interestingly, folate appears able to normalize the homocysteine level independent of the etiology and appears effective regardless of the initial plasma folate levels. Vitamin B12 is

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somewhat less effective than folate in reducing plasma homocysteine (93), and pyridoxine appears useful only in documented deficiency conditions. Recently, several large trials have tested the ability of various vitamin regimens to reduce plasma homocysteine in large populations. Naurath et al. (94) in a prospective double-blinded multicenter trial treated 285 unselected elderly persons (65 to 96 years) for a 3-week period with an extensive intramuscular vitamin regimen including folate, vitamin B12, and pyridoxine. The treatment group demonstrated significant reduction in plasma homocysteine compared to controls irrespective of vitamin levels. Two additional trials (95,96) screened for homocysteinemia in over 300 patients under 55 years of age with symptomatic cerebral or peripheral vascular disease. Approximately one-third of patients demonstrated homocysteine levels $95% percentile. All patients identified were treated with 6 weeks of folate with pyridoxine added in some cases. Of those treated, 90% to 95% normalized at the end of 6 weeks, and the remainder normalized after treatment for an additional 6 weeks. Major medical resources are currently being allocated to the study of homocysteine and atherosclerosis. A recent meta-analysis of currently published data on the topic estimated that up to 50,000 annual coronary deaths may be prevented by dietary and tablet folate supplementation (97). Despite the enthusiasm of the medical community, the most important issue remains unanswered. Will normalization of elevated plasma homocysteine in patients with symptomatic atherosclerosis prove efficacious in preventing and/or arresting the disease process? The next decade will probably provide the answer to this question and hopefully define the role of homocysteine reduction in the prevention/treatment of atherosclerosis.

This work was supported by Grant 1RO1HL45267-01A1, NIH, NHLBI, and by Grant MO1 RR00334, NIH, GCRCB.

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