Importance of Elevated Plasma Homocysteine Levels as a Risk Factor for Atherosclerosis

Importance of Elevated Plasma Homocysteine Levels as a Risk Factor for Atherosclerosis Philippe A. Masser, MD, Lloyd M. Taylor, Jr, MD, and John M. Porter, MD Division of Vascular Surgery, Oregon Health Sciences University, Portland, Oregon Atherosclerosis is a leading cause of death and disability in the Western world, and an important risk factor for it may be an elevated level of the plasma amino acid homocysteine. The biochemical characteristics of homocysteine, along with historical, laboratory, and clinical evidence for its pathologic role in atherosclerosis, are reviewed. Possible therapies for reducing elevated homocysteine levels and the possible impact of therapy in atherosclerosis are examined. (Ann Thorae Surg ) A therosclerosis is the leading cause of death and disability in the nations of the first world. Despite an appropriately massive and ongoing research effort, we have failed to determine either a cause or a cure for this most serious disease. Autopsy findings repeatedly indicate that atherosclerosis is present to some degree in all aged individuals, suggesting that it should perhaps be regarded as a normal result of aging, as well as a distinct disease entity. The distinction between atherosclerosis as a normal consequence of aging and atherosclerosis as a disease resulting in disability and death is important. Atherosclerosis is most appropriately regarded as a disease process when both rapid progression and clinical symptoms occur. The widely accepted risk factors for atherosclerotic disease are age, diabetes, tobacco use, arterial hypertension, hypercholesterolemia, hypertriglyceridemia, decreased high-density lipoprotein cholesterol level, hypercoagulability, sedentary life-style, and elevated plasma homocysteine level. Abnormal lipid values are notable in this list, and the study of lipid metabolism has dominated atherosclerosis research for many years. Despite this, a large number of people with symptomatic atherosclerotic disease have no detectable evidence of abnormal lipid metabolism [1, 2]. An elevation in the plasma homocysteine level, though less familiar to many physicians, is emerging as a recognized risk factor for atherosclerosis. A description of the metabolism of homocysteine, its relationship to vascular disease, and the evidence indicating that homocysteine is an important risk factor for both the presence and rapid progression of atherosclerotic disease form the basis of this review. Homocysteine Metabolism Homocysteine is a sulfur-containing amino acid (Fig 1) that is not one of the 20 amino acids serving as structural components of all proteins. Instead, homocysteine is a Presented at the Cardiovascular Surgery Symposium Honoring Will Camp Sealy, MD, Macon, GA, March 4, 1994. Address reprint requests to Dr Porter, Division of Vascular Surgery, OP-11, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201-3098. 1994 by The Society of Thoracic Surgeons metabolic intermediary, formed by the demethylation of dietary methionine. The reaction of homocysteine with serine, in the presence of the enzyme cystathionine {3-synthase, results in the formation of cystathionine, which is then cleaved to form homoserine plus cysteine. This series of reactions, by which the four-carbon dietary amino acid methionine is converted into the three-carbon amino acid cysteine, is called the transsulfuration pathway. The critical step in this pathway is the formation of cystathionine from homocysteine, as this step is irreversible in human beings. This means that a deficiency of cystathionine {3-synthase, which requires pyridoxine as a vitamin cofactor, results in the abnormal accumulation of homocysteine (Fig 2). Homocysteine can be remethylated to reform methionine by means of reactions that require the enzymes methyltransferase and methylenetetrahydrofolate reductase and the vitamins folate, cobalamin, betaine, or choline. A deficiency of these vitamins or enzymes thus also results in an abnormal accumulation of homocysteine. In plasma, homocysteine exists in three forms: homocysteine, the disulfide homocystine, and the mixed disulfide homocysteine-cysteine (see Fig 1). There is almost no free homocysteine in normal plasma, as nearly all homocysteine in all forms is bound to plasma proteins [3]. History of Homocysteine and Vascular Disease Ignatowsky [4] was the first to demonstrate a relationship between diet and atherosclerosis. He produced atherosclerotic changes in the vessels of rabbits by feeding them meat, milk, and eggs, experiments prompted by his autopsy observations of more prominent atherosclerotic changes in prosperous patients [4]. Newburgh and Clarkson [5] repeated these experiments, demonstrating in 1923 that feeding animals excessive protein produced atherosclerosis. Despite these important findings, the protein theory of atherosclerosis subsequently fell into disfavor and interest shifted to lipid metabolism, a situation essentially unchanged to the present. It was not until 1962 that the inborn error of metabolism, homocystinuria, was described [6, 7]. In this rare disease, homocysteine accumulates in plasma and tissue, and is excreted in large amounts in the urine. Patients have 0003-4975/94/$7.00

Ann Thorae Surg SEALY SYMPOSIUM MASSER ET AL 1241 HS - CH 2 - CH 2 - yh - COOH NH 2 Homocysteine yh 2-CH2 - yh-cooh ~ NH 2 S I CH 2 - CH 2 - yh - COOH NH 2 Homocystine 50 40 30 Normal B Elevated 44 nmol/ml p<.05 p<.05 yh 2-yH-COOH S NH 2 ~ Cysteine I CH - CH -CH - COOH 2 2 I NH 2 Homocysteine 20 10 Mixed Disulfide Fig 1. Forms of homocysteine found in human plasma: homocysteine, homocysteine-cysteine mixed disulfide, and homocystine. multiple abnormalities, including dislocated lens, mental retardation, skeletal disorders, and severe vascular disease. The vascular disease that occurs consists of widespread arterial and venous thrombosis, in addition to virulent arterial intimal proliferation and plaque formation. Fatal arterial thrombosis usually occurs early in life [8]. Homocystinuria is most frequently caused by a homozygous deficiency of the enzyme cystathionine {3-synthase [9, 10]. Patients with typical homocystinuria, including vascular disease, who are deficient in methyltransferase [II] and methylenetetrahydrofolate reductase [12] have also been identified. The occurrence of the same clinical syndrome in patients with different enzyme deficiencies, all of which result in the abnormal accumulation of homocysteine, provided strong circumstantial evidence that homocysteine itself was the toxic substance. The infusion of homocysteine into laboratory animals confirmed the rapid formation of typical vascular lesions [13]. Interestingly, the treatment of homocystinemic animals and humans with pyridoxine or folic acid, or both, ameliorated some of the laboratory and clinical manifestations of homocystinemia [13-16]. These findings suggested an obvious reason for investigating the possible role of abnormalities in homocysteine metabolism in the genesis of atherosclerosis and its progression to symptomatic clinical disease. N 5 Methyl TetrahYdrOfOl8t( t N 5,10 Methylene Tetrahydrofolate C H 3 S C ~ C H 2 C H (NH 2)COOH I Methionine / SCH 2CH2CH(NH2)COOH Homocystine 8 2 8 12 t HSCH 2CH 2CH (NH 2 icooa Homocysteine 0)1 + HOCH 2CH(NH2)COOH +8 6 Serine SCH 2CH(NH2)COOH CH2CH2CH(NH2)COOH Cystathionine ~ C H 2 C H 2 C H ( N H 2 ) C O O H ~HOCH 2CH 2CH(NH2)COOH Homosertne + HSCH 2CH (NH 2)COOH Cysteine Fig 2. Metabolism of homocysteine: Enzyme 1 is cystathionine (3-synthase; enzyme 2 is homocysteine methyltransferase; enzyme 3 is methylenetetrahydrofolate reductase. Basal After Methionine load After Food load Fig 3. Plasma total homocysteine levels in 13 subjects before and after methionine loading, and before and after meals. Numbers at the top of the bars are the mean plasma homocysteine values in nanomoles per microliter. (Reprinted from Taylor LM [r, Porter JM. Elevated plasma homocysteine as a risk factor for atherosclerosis. Semin Vase Surg 1993;6:36-45, by permission of W.B. Saunders.) Clinical Studies Relating Homocysteine to Atherosclerosis Traditional amino acid analysis is sufficiently insensitive as to be unable to detect the minute amounts of homocysteine present in normal plasma. Initial studies of the relationship of plasma homocysteine to atherosclerotic disease therefore relied on methionine loading tests. In these tests, the administration of large amounts of methionine in subjects results in an increase in the plasma homocysteine concentration to detectable levels, with the magnitude of the increase used to differentiate normal from abnormal subjects [17, 18]. An abnormal response to methionine loading identifies subjects heterozygous for cysthionine {3-synthase deficiency, a condition calculated to occur in between 1 in 70 and 1 in 200 persons. Measurement of the total plasma homocysteine level, combined with the more sensitive method highperformance liquid chromatography, allows normal levels to be differentiated from abnormal levels without the need for either loading or tolerance studies. In our own laboratory, we showed that all subjects tested responded to oral methionine loading with an increase in the plasma homocysteine level, and that the resultant increase was proportionate to the basal levels of homocysteine. Thus, patients with elevated basal levels of homocysteine uniformly showed markedly elevated levels in response to methionine loading. We also found that testing in the fasting state was unnecessary, an important advantage for population screening studies (Fig 3). Others have also observed this [3]. These findings mean that plasma homocysteine testing can be performed at the time of any patient visit, and is very capable of distinguishing between normal and abnormal results. At present, there is no universally accepted method of plasma homocysteine testing. Many investigators use highperformance liquid chromatography, but there are multiple techniques, types of detectors, and so on [19-24]. Other methods in use include gas chromatography-mass spectrometry [25] and radioenzymatic assay [26]. Because of

1242 SEALY SYMPOSIUM MASSER ET AL Ann Thorac Surg these differences, there is no generally accepted range of normal values. Most laboratories have found that the plasma homocysteine levels in men are slightly higher than those in women, and that the levels in premenopausal women are slightly lower levels than those in postmenopausal women [27]. The ranges of normal values determined in multiple studies using different detection techniques are listed in Table 1. It is important to note, however, that, to date, all studies have determined normal values based on control populations of asymptomatic individuals. These persons were presumed to have no atherosclerosis based on their lack of symptoms rather than on any objective proof of an absence of atherosclerotic lesions. Findings from studies performed in fraternal and identical twins suggest that homocysteine levels are genetically influenced [34]. Multiple other factors have been shown to result in an abnormal elevation of plasma homocysteine values. Decreased levels of folate [27,35], pyridoxine [36], or vitamin B 12 [27, 37, 38] can cause plasma homocysteine levels to be elevated [39]. The plasma homocysteine concentration is elevated in an inverse relationship to renal function, with high levels consistently found in patients undergoing hemodialysis [40-42]. Several studies have revealed relationships between elevated plasma homocysteine levels, elevated uric acid values, and diuretic use [3, 32,43]. This is probably explained by the strong influence of decreased renal function in elevating the plasma homocysteine level. In a considerable number of studies, a consistent relationship has been noted between an elevated plasma homocysteine concentration and the presence of athero- Table 1. Normal Plasma Homocysteine Values Author Israelsson et al [29] Araki et al [30] Genest et al [31] Coull et al [32] Jacobsen et al [33] a Numbers in parenthesis are the number of patients. Asymp = asymptomatic. Subjects" Malinkow et al [19] Asymp men <60 y (35) Asymp men >60 y (18) Asymp women <60 y (39) Asymp women >60 y (11) Jacobsen et al [27] Asymp men, mean age 34 y (36) Asymp women, mean age 34 y (35) Brattstrom et al [28] Asymp men, mean age 61 y (34) Asymp women, mean age 61 y (32) Male controls (36) Asymp controls (both sexes) (45) Hypertensive controls (45) Framingham asymp males (255) Asymp both sexes, mean age 61 y (31) Males, mean age 39 y (12) Females, mean age 37 y (12) Homocysteine (flmol/l) 11.18 ± 3.58 10.74 ± 2.16 8.58 ± 2.82 9.04 ± 2.16 9.26 ± 1.88 7.85 ± 2.29 12.7 ± 2.5 11.1 ± 3.6 13.5 ± 3.6 7.3 ± 2.9 9.9 ± 4.1 10.9 ± 4.9 10.68 ± 3.20 15.8 ± 6.4 16.5 ± 4.4 Table 2. Studies Confirming Elevated Plasma Homocysteine Levels as a Risk Factor for Arterial Disease 0/0 With Elevated Ref No., Patient Control Homocysteine Year Patients" Mean" Mean b Level [181, 1985 Peripheral artery 16.1 10.1 47 disease (47) [31, 1986 Coronary artery 5.48 4.25 15 disease (241) [291, 1988 Myocardial 16.4 13.5 24 infarction (21) [301, 1989 Stroke (45) 13.1 7.3 NG [441, 1990 Coronary artery 13.07 11.26 19 disease (64) [321, 1990 Stroke (41) 15.78 10.68 NG [311, 1991 Coronary artery 13.9 10.9 28 disease (176) [431, 1991 Cerebral + 14.3 10.1 39 peripheral disease (214) [451, 1992 Claudication (78) 16.74 13.8 23 [281, 1992 Stroke (142) 18.6 11.9 40 [461, 1993 Diabetic 10.8 7.5 NG macroangiopathy (52) a Numbers in parenthesis are numbers of patients. given as micromoles per liter of homocysteine. NG = value not given. b All values are sclerotic disease in the coronary, cerebral, and peripheral arterial circulations. The results of multiple representative studies are summarized in Table 2. The mean plasma homocysteine values in these studies have averaged about 25% to 50% higher in patients with atherosclerotic disease than in controls. When examined individually, most series have shown that 15% to 40% of patients with disease have plasma homocysteine values considerably higher than control mean values. All studies to date have shown that an elevation in the plasma homocysteine concentration occurs independently of the other recognized risk factors for atherosclerotic disease noted earlier. This laboratory finding cannot be explained as a cofinding stemming from tobacco use, diabetes, abnormal lipid levels, or advanced age [3, 19, 29-32, 43-46]. To date, only a few studies have examined whether abnormally high homocysteine values found in patients with symptomatic atherosclerosis can be explained by corresponding abnormalities in the known cofactors for homocysteine metabolism, such as folate or vitamins B 6 and B 1 2, or by abnormal renal function. Molgaard and co-workers [45] found that the plasma homocysteine level was elevated almost entirely in patients with serum folate levels less than 11.0 nmol/l. In the study conducted by Brattstrom and associates [28], using multiple regression analysis, they found that about 40% of the variability in the plasma homocysteine levels could be explained by a corresponding variability in age, cofactor concentrations, and serum creatinine levels. Lewis and colleagues [47], in their

Ann Thorae Surg SEALY SYMPOSIUM MASSER ET AL 1243 Elevated H(e) 100 p<o.ol 90 f]j Normal H(e) 80 70 c: 60 Q) o., 50 a. 40 30 20 10 0 Clin Prog of Clin Clin Clin Vase Vase Disease (all LED CVD CAD Lab LED Lab CVD categories) Prog Prog Prog Prog Prog Fig 4. Presence of clinical and laboratory evidence of progression of disease in patients with normal and elevated plasma homocysteine levels. (CAD = coronary artery disease; Clin = clinical; CVD = carotid artery disease; Hte) = homocysteine; LED = lower extremity disease; Prog = progression; Vase = tiascular.) The percent axis refers to the percentage of all patients in each category showing evidence of disease progression. (Reprinted from Taylor LM Jr, Porter JM. Elevated plasma homocysteine as a risk factor for atherosclerosis. Semin Vasc Surg 1993;6:36-45, by permission of W.B. Saunders.) study comparing patients with coronary artery disease to controls, concluded that folate levels higher than those generally accepted as normal were necessary to prevent plasma homocysteine levels from becoming elevated. Because of evidence that at least some hyperhomocystinemia results from inherited enzyme deficiencies, it is of considerable interest to know what proportion of the cases of hyperhomocystinemia result from this mechanism. In the study conducted by Genest and associates [31), plasma homocysteine testing was performed on family members of patients with coronary artery disease who had elevated plasma homocysteine values (28% of the patients). A familial association was noted for half of the patients with elevated values (14% of all patients). Thus, an important percentage of patients with symptomatic atherosclerotic disease have elevated plasma homocysteine levels, and this elevation may result from multiple causes, including cofactor deficiencies, renal insufficiency, and inherited enzyme deficiencies. Speculation has also centered on whether an elevated plasma homocysteine concentration is a marker for the more rapid progression of symptomatic atherosclerotic disease. To date, only a single retrospective study has addressed this issue [43]. Patients with elevated plasma homocysteine levels were found to be significantly more likely to experience clinical events indicating progression of lower extremity and coronary arterial disease, as well as to have vascular laboratorydetermined evidence of progression of lower extremity arterial disease, than were patients with normal plasma homocysteine levels (Fig 4). In addition, the rate of clinical progression of disease was more rapid in patients with elevated plasma homocysteine levels (Fig 5). Multiple regression analysis indicated that none of these differences was explained by an association of elevated plasma homocysteine levels with other known clinical risk factors for atherosclerosis. In a case-control study, elevated homocys- teine levels were found to be associated with an increased likelihood of carotid artery intimal-medial wall thickness in asymptomatic adults [48]. In a recent study, plasma homocysteine testing was performed on blood samples obtained prospectively during the ongoing Physicians Health Study. These investigators found the relative risk of myocardial infarction for the men with the highest 5% of plasma homocysteine values was 3.1 [49]. This important study is the first to identify an elevated plasma homocysteine concentration as a significant prospective risk factor for atherosclerotic diseaserelated events. Laboratory Evidence Relating Homocysteine Levels to Atherosclerosis The clinical studies described in the preceding section have yielded considerable evidence for an association between an elevation in the plasma homocysteine concentration and symptomatic atherosclerosis. Animal experiments have demonstrated that typical vascular lesions are rapidly produced after the infusion of homocysteine [13]. These studies were important because they showed homocysteine itself had a direct toxic effect. Since then, multiple studies have shown the existence of possible mechanisms by which homocysteine may interact with blood vessels or the clotting system, or both, to cause symptomatic vascular disease. McCully demonstrated that homocysteine has a direct toxic effect on cultured endothelial cells [50] and that, in experimental models of atherogenesis, an elevated plasma homocysteine concentration acts in concert with dietary lipids [51]. He theorized that excess amounts of homocysteine result in the abnormal sulfation of proteoglycans through the intermediary substance homocysteine thiolactone, a known cellular toxin [52, 53]. Others have suggested that the vascular endothelium in patients with Elevated H(e) 0.50 f]j Normal H(e) 0.45 p < 0.002 0.40 0.35 0.30 ll.. 0.25 o 0.20 0.15 0.10 0.05 0.00 Clinical Progresion Rate Fig 5. Rate of clinical progression of disease (lower extremity plus carotid artery plus coronary artery disease) in patients with elevated plasma homocysteine levels compared to the rate in patients with a normal plasma homocysteine level. (CPI = clinical progression index, calculated by dividing the duration of clinical follow-up in months by the number of clinical progression events; H(e) = homocysteine.) (Reprinted from Taylor LM [r, Porter JM. Elevated plasma homocysteine as a risk factor for atherosclerosis. Semin Vasc Surg 1993;6:36-45, by permission of W.B. Saunders.)

1244 SEALY SYMPOSIUM MASSER ET AL Ann Thome Surg cystathionine l3-synthase deficiency is more susceptible to homocysteine-induced injury [54] and that homocysteine injures the endothelial cells through the generation of hydrogen peroxide [55,56]. In vitro studies involving the use of cultured endothelial cells have demonstrated possible links among homocysteine, thrombosis, and atherogenesis. Investigators have shown that the addition of homocysteine to culture media can reduce normal activation of protein C by endothelial cells [57], increase the binding of lipoprotein (a) to plasmin-modified fibrin [58], and induce tissue factor procoagulant activity [59]. Homocysteine inhibits the cofactor activity of thrombomodulin, thus reducing the antithrombotic property of endothelial cells [60]. Elevated homocysteine levels may cause the expression of other cell surface proteins to be reduced as well [61]. Selhub and Miller [62] have recently hypothesized that S-adenosyl-methionine regulates the partitioning of cellular homocysteine between being degraded to cystathionine and remethylated to methionine, and that this regulation is impaired in the setting of homocysteinemia. They believe this may explain why homocysteine levels increase when one metabolic pathway is blocked rather than remaining unchanged because of diversion to the other pathway. Though the importance of any of these diverse mechanisms in the human form of the disease is unknown, each may have an important role in the influence of elevated homocysteine levels on vascular disorders. Implications of Homocysteine as a Risk Factor for Atherosclerosis With Reference to Prevention and Treatment The ultimate goal of all risk factor research is to identify useful methods of disease prevention and treatment. With respect to plasma homocysteine levels there is intriguing evidence that it may be possible to identify methods for reducing these levels that are simple, nontoxic, and inexpensive. At present, it is known that the favorable modification of some risk factors for atherosclerotic disease results in an improved prognosis. Examples include modification of tobacco use, hypercholesterolemia, hypertension, and, to a lesser degree, a sedentary life-style. The modification of any of these factors requires radical changes in addictions or behavior, or both, or the use of medications with considerable side effects. Because of this, success rates for the modification of atherosclerotic risk factors are low. The clear-cut relationship between the plasma homocysteine levels and vitamin cofactors means that elevated levels can be reliably reduced to normal in most patients by the administration of folate [63, 64]. Those whose elevated homocysteine levels are resistant to folate treatment usually respond to treatment with nontoxic doses of pyridoxine, vitamin B 12, choline, or betaine [65-68]. Folate appears to be able to lower the plasma homocysteine concentration independent of the cause of the hyperhomocystinemia, in that folate treatment is effective in patients without folate deficiency [28]. Vitamin B 12 therapy alone has been less reliably effective in lowering homocysteine levels [46,69], and pyridoxine treatment is probably effec- tive only in the deficiency state or in combination with folate supplementation [39]. Thus, if lowering the elevated plasma homocysteine levels in patients with symptomatic atherosclerosis is associated with a clinical benefit, this could be the first treatment for atherosclerosis that is effective and free of side effects and does not require changes in basic life-style or habits. The present state of our knowledge puts us considerably short of this goal. We currently do not know whether atherosclerotic patients with elevated homocysteine levels have more severe or rapidly progressive disease than do those with normal levels, and we have no evidence suggesting that the therapeutic lowering of high levels confess a clinical benefit. Prospective studies to examine this are needed, and several are currently under way. The next 4 to 5 years should witness important increases in our knowledge of what may be a very important piece in the puzzle of atherosclerotic disease. Supported by grant 1R01HL45267-01A1, NIH, NHLBI, and grant Mal RR00334, NIH, GCRCB. References 1. Gordon T, Garcia-Palmieri MR, Dagan A, et al. Differences in coronary heart disease in Framingham, Honolulu, and Puerto Rico. J Chronic Dis 1974;27:329-44. 2. McCully KS. Atherosclerosis, serum cholesterol and the homocysteine theory: a study of 194 consecutive autopsies. Am J Med Sci 1990;299:217-21. 3. Kang SS, Wong PWK, Cook HY, et al. Protein-bound homocyst(e)ine: a possible risk factor for coronary artery disease. J Clin Invest 1986;77:1482-6. 4. Ignatowsky IA. Influence de la nourriture animale sur l'organisme des lapins. Arch Med Exp Anat PathoI1908;20:1 20. 5. Newburgh LH, Clarkson S. The production of atherosclerosis in rabbits by feeding diets rich in meat. Arch Intern Med 1923;31:653-76. 6. Carson NAJ, Neill DWS. Metabolic abnormalities detected in a survey of mentally backward individuals in Northern Ireland. Arch Dis Child 1962;37:505-13. 7. Gerritsent T, Vaughn JG, Waisman HA. The identification of homocystine in the urine. Biochem Biophys Res Commun 1962;9:493-6. 8. Mudd SH, Skovby F, Levy HL, et al. The natural history of homocystinuria due to cystathionine (3-synthase deficiency. Am J Hum Genet 1985;37:1-31. 9. Mudd SH, Finkelstein JD, Irrevere F, Laster L. Homocystinuria: an enzymatic defect. Science 1964;143:1443-5. 10. Hu FL, Gu Z, Kozich V, Kraus JP, Ramesh V, Shih VE. Molecular basis of cystathionine (3-synthase deficiency in pyridoxine responsive and nonresponsive homocystinuria. Hum Mol Genet 1993;2:1857-60. 11. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111-28. 12. Mudd SH, Uhlendorf BW, Freeman JM, Finklestein JD, Shih VB. Homocystinuria associated with decreased methylenetetrahydrofolate reductase activity. Biochem Biophys Res Commun 1972;46:905-12. 13. Harker LA, Slichter SF, Scott CR, Ross R. Homocystinemia: vascular injury and arterial thrombosis. N Engl J Med 1974; 291:537-43. 14. Harker LA, Ross R, Slichter SF, Scott CR. Homocystineinduced arteriosclerosis. J Clin Invest 1976;16:731-41. 15. Hladovec J. Experimental homocystinemia, endothelial lesions and thrombosis. Blood Vessels 1979;16:202-5.

Ann Thorae Surg SEALY SYMPOSIUM MASSER ET AL 1245 16. Hladovec J. Methionine, pyridoxine and endothelial lesions in rats. Blood Vessels 1980;17:104-9. 17. Wilcken DEL, Wilcken B. The pathogenesis of coronary artery disease: a possible role for methionine metabolism. J Clin Invest 1976;57:1079-82. 18. Boers GHJ, Smals AGH, Trijbels FJM, et al. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 1985;313:709-15. 19. Malinow MR, Kang SS, Taylor LM [r, et al. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation 1989;79:1180-8. 20. Smolin LA, Schneider JA. Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection. Anal Biochem 1988;168:374-9. 21. Brattstrom LE. Heterozygosity for homocystinuria in premature arterial disease. N Engl J Med 1986;314:849-50. 22. Cornwell PE, Morgan SL, Vaughn WHo Modification of a high-performance liquid chromatographic method for assay of homocysteine in human plasma. J Chromatogr 1993;617: 136-9. 23. Andersson A, Isaksson A, Brattstrom L, Hultberg B. Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization. Clin Chern 1993;39: 1590-7. 24. Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chern 1993;39: 263-71. 25. Stabler SP, Marcell PO, Podell ER, Allen RH. Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Biochem 1987;162:185-96. 26. Refsum H, Helland S, Ueland PM. Radioenzymic determination of homocysteine in plasma and urine. Clin Chern 1985; 31:624-8. 27. Jacobsen OW, Gatautis VJ, Green R, et al. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate levels in normal subjects. Clin Chern 1994;40: 873-81. 28. Brattstrom L, Lindgren A, Israelsson B, Malinow MR, Norrving B, Upson B. Hyperhomocysteinaemia in stroke: prevalence, cause, and relationship to type of stroke and stroke risk factors. Eur J Clin Invest 1992;22:214-21. 29. Israelsson B, Brattstrom LE, Hultberg BL. Homocysteine and myocardial infarction. Atherosclerosis 1988;71:227-33. 30. Araki A, Sako Y, Fukushima Y, et al. Plasma sulfhydryl-containing amino acids in patients with cerebral infarction and in hypertensive patients. Atherosclerosis 1989;79:139-46. 31. Genest JJ [r, McNamara JR, Upson B, et al. Prevalence of familial hyperhomocyst(e)inemia in men with premature coronary artery disease. Arterioscler Thromb 1991;11:1129-36. 32. Coull BM, Malinow MR, Beamer N, Sexton G, Nordt F, degarmo P. Elevated plasma homocyst(e)ine concentration as a possible independent risk factor for stroke. Stroke 1990;21: 572-76. 33. Jacobsen OW, Gatautis VJ, Green R. Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection. Anal Biochem 1989;178:208-14. 34. Reed T, Malinow MR, Christian [C, Upson B. Estimates of heritability of plasma homocyst(e)ine levels in aging adult male twins. Clin Genet 1991;39:425-28. 35. Kang SS, Wong PWK Norusis M. Homocysteinemia due to folate deficiency. Metabolism 1987;36:458-62. 36. Smolin LA, Crenshaw TO, Kurtyca 0, Benevenga NJ. Homocyst(e)ine accumulation in pigs fed diets deficient in vitamin B6: relationship to atherosclerosis. J Nutr 1983;133:2022-33. 37. Brattstrom L, Israelsson B, Lindgarde F, Hultberg B. Higher total plasma homocysteine in vitamin B12 deficiency than in heterozygosity for homocystinuria due to cystathionine {3-synthase deficiency. Metabolism 1988;37:175-8. 38. Joosten E, Pelemans W, Devos P, et al. Cobalamin absorption and serum homocysteine and methylmalonic acid in elderly subjects with low serum cobalamin. Eur J Haematol 1993;51: 25-30. 39. Mason JB, Miller JW. The effects of vitamins B12, B6, and folate on blood homocysteine levels. Ann NY Acad Sci 1992;669:197 203. 40. Wilcken DEL, Gupta VJ. Sulphur containing amino acids in chronic renal failure with particular reference to homocysteine and cysteine-homocysteine mixed disulphide. Eur J Clin Invest 1979;9:301-7. 41. Kang SS, Wong PWK, Bidani A, Milanez S. Plasma proteinbound homocyst(e)ine in patients requiring chronic haemodialysis. Clin Sci 1983;65:335-6. 42. Chauveau P, Chadefaux B, Coude M, et al. Increased plasma homocysteine concentration in patients with chronic renal failure. Miner Electrolyte Metab 1992;18:196-8. 43. Taylor LM Jr, DeFrang RD, Harris EJ Jr, Porter JM. The association of elevated plasma homocyst(e)ine with progression of symptomatic peripheral arterial disease. J Vase Surg 1991;13:128-36. 44. Malinow MR, Sexton G, Averbuch M, Grossman M, Wilson 0, Upson B. Homocyst(e)inemia in daily practice: levels in coronary artery disease. Coronary Art Dis 1990;1:215-20. 45. Molgaard J, Malinow MR, Lassvik C, Holm AC, Upson B, Olsson AG. Hyperhomocyst(e)inaemia: an independent risk factor for claudication. J Intern Med 1992;231:273-9. 46. Araki A, Sako Y, Ito H. Plasma homocysteine concentrations in Japanese patients with non-insulin-dependent diabetes mellitus: effect of parenteral methylcobalamin treatment. Atherosclerosis 1993;103:149-57. 47. Lewis CA, Pancharuniti N, Sauberlich HE. Plasma folate adequacy as determined by homocysteine level. Ann NY Acad Sci 1992;669:360-2. 48. Malinow MR, Nieto FJ, Szklo M, Chambless LE, Bond G. Carotid artery intimal-medial wall thickening and plasma homocyst(e)ine in asymptomatic adults. The Atherosclerosis Risk in Communities Study. Circulation 1993;87:1107-13. 49. Stampfer MJ, Malinow MR, Willett Wc, et al. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 1992;268:877-81. 50. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of atherosclerosis. Am J Pathol 1969;56:111-28. 51. McCully KS, Olszewski AJ, Vezeridis MP. Homocysteine and lipid metabolism in atherogenesis; effect of the homocysteine thilactonyl derivatives, thioretinaco and thioretinamide. Atherosclerosis 1990;83:197-206. 52. McCully KS. Homocysteine theory of arteriosclerosis: development and current status. In: Gotto AM Ir, Paoletti R, eds. Atherosclerosis reviews. New York: Raven P, 1983;11:157-247. 53. Dudman NPB, Hicks C, Lynch JF, Wilken DEL, Wang J. Homocysteine thiolactone disposal by human arterial endothelial cells and serum in vitro. Arterioscler Thromb 1991;11: 663-70. 54. Wang J, Dudman NPB, Wilcken DEL, Lynch JF. Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease. Atherosclerosis 1992;97:97-106. 55. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 1986;77:1370-6. 56. Berman RS, Martin W. Arterial endothelial barrier dysfunction: actions of homocysteine and the hypoxanthine-xanthine oxidase free radical generating system. Br J Pharmacol 1993; 108:920-6. 57. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood 1990;75:895-901. 58. Harpel PC, Chang VT, Borth W. Homocysteine enhances the binding of lipoprotein (a) to plasmin-modified fibrin providing a potential link between thrombosis and atherogenesis [abstract]. Blood 1990;76(suppl 1):510a. 59. Fryer RH, Wilson BD, Gubler DB, Fitzgerald LA, Rodgers GM. Homocysteine, a risk factor for premature vascular disease

1246 SEALY SYMPOSIUM MASSER ET AL Ann Thorae Surg and thrombosis, induces tissue factor activity in endothelial cells. Arterioscler Thromb 1993;13:1327-33. 60. Hayashi T, Honda G, Suzuki K. An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombomodulin expression in human umbilical vein endothelial cells. Blood 1992;79:2930-6. 61. Lentz SR, Sadler JE. Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum. Blood 1993;81:683-9. 62. Selhub I, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 1992;55:131-8. 63. Brattstrom LE, Israelsson B, Jeppsson JO, Hultberg BL. Folic acid-an innocuous means to reduce plasma homocysteine. Scand J Clin Lab Invest 1988;48:215-21. 64. Arnadottir M, Brattstrom L, Simonsen 0, et al. The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients. Clin Nephrol 1993;40:236-40. 65. Wilcken DEL, Wilcken B, Dudman NPB, Tyrrell PA. Homocystinuria-the effects of betaine in the treatment of patients not responsive to pyridoxine. N Engl J Med 1983;309:448-53. 66. Stabler SP, Marcell PO, Podell ER, et al. Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatographymass spectrometry. J Clin Invest 1988;81:466-74. 67. Olszewski AI, Szostak WB, Bialkowska M, et al. Reduction of plasma lipid and homocysteine levels by pyridoxine, folate, cobalamin, choline, riboflavin, and troxerutin in atherosclerosis. Atherosclerosis 1989;75:1-6. 68. Wilcken DEL, Dudman NPB, Tyrrell PA, Robertson MR. Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: possible implications for prevention of vascular disease. Metabolism 1988;37:697-701. 69. Joosten E, Pelemans W, Devos P, et al. Cobalamin absorption and serum homocysteine and methylmalonic acid in elderly subjects with low serum cobalamin. Eur J Haematol 1993;51: 25-30.