Homocysteinemia as a risk factor for atherosclerosis: a review

PII: S0967-2109(97)00062-8

Cardiovascular Surgery, Vol. 5, No. 6, pp. 559–567, 1997  1997 The International Society for Cardiovascular Surgery Published by Elsevier Science Ltd. Printed in Great Britain 0967–2109/97 $17.00 + 0.00

VASCULAR REVIEW Homocysteinemia as a risk factor for atherosclerosis: a review M. R. Nehler, L. M. Taylor Jr and J. M. Porter Department of Surgery, Division of Vascular Surgery, Oregon Health Sciences University, Portland, Oregon, USA In industrialized nations, the leading cause of death and disability is atherosclerosis. 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 show atherosclerosis to some degree in nearly all aged people, suggesting it should be regarded as a normal aging process as well as a disease. Any investigation of atherosclerosis etiology therefore requires a distinction between atherosclerosis normally observed with aging, and pathologic atherosclerosis causing disease 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, some hypercoaguable states, sedentary life-style, and elevated plasma homocysteine. The study of lipid metabolism has dominated research into atherosclerosis etiology for decades, although now it is widely recognized that a large number of people with symptomatic atherosclerotic disease have no detectable evidence of abnormal lipid metabolism [1, 2]. Elevation in plasma homocysteine has also 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 the potential role of treatment will form the basis for this review.  1997 The International Society for Cardiovascular Surgery Keywords: atherosclerosis, etiology, homocysteine, vascular disease, treatment

Homocysteine metabolism Homocysteine is a sulfur-containing amino acid not included in the 20 amino acids that serve as protein structural elements. Three forms exist in human plasma: homocysteine, the disulfide homocystine, and the mixed disulfide homocysteine–cysteine (Figure 1). Almost all plasma homocysteine is bound to proteins. Homocysteine functions as a metabolic

Correspondence to: Dr L. M. Taylor Jr, Department of Surgery, Division of Vascular Surgery (OP-11), Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 972013098, USA

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intermediary following either the demethylation of dietary methionine during the formation of cysteine, or during re-methylation to form methionine (Figure 2). In the former, the enzyme cystathionine β-synthase with pyridoxine as a vitamin cofactor catalyzes the reaction of homocysteine with serine to form cystathionine, which is then split to form cysteine. This sequence of reactions resulting in the conversion of the four-carbon dietary amino acid methionine to the three-carbon amino acid cysteine is called the transsulfuration pathway. A deficiency of the enzyme cystathionine β-synthase results in abnormal accumulation of homocysteine. Homocysteine may also be re-methylated to form methionine via the enzymes methyltransferase and 559

Homocysteinemia: a risk factor for atherosclerosis: M. R. Nehler et al.

to different enzymatic defects implicates homocysteine as the causative agent. This hypothesis is also supported by animal models demonstrating vascular lesions following homocysteine infusion [11]. Serum homocysteine levels can be lowered in homocysteinemic patients with oral pyridoxine, folic acid, or both [11–13] The potential for relatively simple and effective treatment of this risk factor for atherosclerosis stimulates the current research enthusiasm.

Clinical data Figure 1 Forms of homocysteine found in human plasma: homocysteine, homocysteine-cysteine mixed disulfide and homocystine. (Used with permission from Masser, P.A., Taylor, L.M. Jr and Porter, J.M., Importance of elevated plasma homocysteine as a risk factor for atherosclerosis. Annals of Thoracic Surgery, 1994, 58, 1240–1246.)

methylene-tetrahydrofolate reductase. This step requires donation of a methyl group from cofactors folate, cobalamin, betaine or choline. Deficiencies in any of these enzymes or cofactors may result in an abnormal elevation of plasma homocysteine. These pathways are collectively referred to as the remethylation pathway. The concentration of the intermediary S-adenosyl methionine acts to regulate the intracellular metabolism of homocysteine, including both degradation to cysteine or re-methylation to methionine through inhibition of methylene-tetrahydrofolate reductase and stimulation of cystathionine ß-synthase [3]. A defect in this regulation has been speculated to be responsible for the elevation in homocysteine observed when only one enzymatic limb of homocysteine metabolism is deficient.

Homocysteine and vascular disease The rare inborn error of metabolism, homocystinuria, was first described in 1962 [4, 5]. Homocysteine accumulates in the plasma and tissues, spilling into the urine in large quantities. Afflicted patients have multiple developmental abnormalities, including dislocated lens, mental retardation, and skeletal disorders, in addition to severe premature vascular disease. The vascular manifestations include widespread arterial and venous thrombosis usually resulting in death as early as the first decade [6]. A homozygous defect in the enzyme cystathionine βsynthase is the most frequent etiology [7, 8], but 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 560

Early studies of plasma from patients with homocystinuria described 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 early technical problems with detection sensitivity, multiple clinical evaluations have measured homocysteine levels in response to a oral methionine load to determine the relationship between plasma homocysteine and symptomatic atherosclerosis [18, 19]. An abnormal response to a methionine load (defined as ⱖ 90th percentile of normal plasma homocysteine) identifies patients with significant homocysteinemia. Presently, total plasma homocysteine levels are measured using high-performance liquid chromatography (HPLC) permitting detection of abnormal levels without the need for methionine loading, thus significantly simplifying patient screening evaluation. Our laboratory has demonstrated an increase in patient plasma homocysteine with methionine loading which is proportional to their baseline level. This reproducibility of plasma homocysteine regardless of normal dietary intake eliminates the need for fasting [20], a finding reproduced by others [21]. At present, there is no universally accepted standard assay technique for plasma homocysteine. Many investigators use HPLC, but with a variety of technical differences [22–32]. Accordingly, no accepted range for normal plasma homocysteine has been defined. There is general inter-laboratory agreement that men have higher values than women, and homocysteine levels in postmenopausal women are higher than those in premenopausal women [30, 33, 34]. Various normal values determined in multiple investigations are listed in Table 1. To date, normal values have been based on control populations defined by their symptom free clinical status, without more detailed non-invasive studies to exclude occult atherosclerotic lesions. Another confounding factor in homocysteine research is the large range of conditions demonstrated potentially to affect plasma homocysteine levels. Studies of fraternal and identical twins suggest that homocysteine levels may be genetically influCARDIOVASCULAR SURGERY

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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) methylene tetrahydrofolate reductase; (2) homocysteine methyltransferase; (4) choline dehydrogenase; (6) betaine methyltransferase; (5) cystathionine β-synthase; (9) γ-cystathionase: THF, tetrahydrofolate, PLP pyridoxal-5⬘-phosphate. *Areas in the pathway with vitamin cofactors. (Used with permission from Selhub, J. and Miller, J.W., 1992 [3].

enced [35]. Deficiencies in the cofactors 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 CARDIOVASCULAR SURGERY

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the excess burden of cardiovascular disease observed in the end-stage renal disease population [48]. Evidence exists for hormonal regulation of plasma homocysteine in women. As noted above, postmenopausal women have higher plasma homocysteine levels than premenopausal women. 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]. 561

Homocysteinemia: a risk factor for atherosclerosis: M. R. Nehler et al. Table 1 Normal plasma homocysteine values Subjects (n)

Mean (SD) Homocysteine

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

11.2 10.7 8.6 9.0 9.3 7.9 12.7 11.1 13.5 7.3 9.9 10.9 10.7 15.8 16.5

References

(3.6) (2.1) (2.8) (2.2) (1.9) (2.3) (2.5) (3.6) (3.6) (2.9) (4.1) (4.9) (3.2) (6.4) (4.4)

[22] [22] [22] [22] [30] [30] [34] [34] [55] [60] [60] [59] [46] [32] [32]

*All values given as µmol/l homocysteine.

Abundant data indicate a consistent relationship between plasma homocysteine and symptomatic atherosclerotic disease involving the coronary, peripheral and cerebral circulations [33, 34, 46, 47, 54– 62]. The results of a number of these studies are shown in Table 2. Mean plasma homocysteine values have been 25–50% higher in patients with symptomatic atherosclerotic disease compared with controls. In addition, most series demonstrate a subset including 15 to 40% of the symptomatic patients with markedly elevated plasma homocysteine ( ⱖ 90 to 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 cofactors of

homocysteine metabolism; folate, vitamins B6 and B12. Molgaard et al. [58] found elevated plasma homocysteine levels occurred almost exclusively in individuals 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. [63] demonstrated that folate levels higher than normal values were necessary to prevent elevated plasma homocysteine in patients with coronary artery disease. A recent study of 75 patients undergoing maintenance hemodialysis found only folate level and renal function to correlate with fasting homocysteine and also suggested that these patients may require supranormal levels of folate to prevent elevated plasma homocysteine [64]. Homocysteine and vitamin levels were examined in 1160 patients from the Framingham study [65]. Some 33% of these patients

Table 2 Studies confirming elevated plasma homocysteine as a risk factor for arterial disease Patients (n)

Peripheral artery disease (47) Myocardial infarct (21) Stroke (45) Coronary disease (64) Stroke (41) Coronary disease (176) Cerebral + peripheral (214) Claudication (78) Stroke (142) Diabetic microangiopathy (52) Coronary artery disease (266) Coronary artery disease (68)

Patient Mean*

16.1 16.4 13.1 13.1 15.8 13.9 14.3 16.7 18.6 10.8 12.0 11.5

Control Mean*

10.1 13.5 7.3 11.3 10.7 10.9 10.1 13.8 11.9 7.5 10.1 14.5

% with elevated homocysteine 47 24 NG 19 NG 28 39 23 40 NG 17.6 22

Year

Reference

1985 1988 1989 1990 1990 1991 1991 1992 1992 1993 1994 1996

[56] [54] [60] [57] [46] [59] [47] [58] [34] [33] [61] [62]

*All values given as µmol/l of homocysteine. NG, not given.

562

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had homocysteine levels above the 90th percentile, and two-thirds of these could be explained by relative vitamin deficiency. A recent review of over 5000 participants in a Canadian nutrition survey identified serum folate deficiency as a significant risk factor for a subsequent cardiac death [66]. 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 5–7% [67, 68]. Homozygous or heterozygous conditions for a thermolabile defect in methylene-tetrahydrofolate reductase [69] or heterozygous cystathionine β-synthase deficiencies are the most frequently observed genetic causes. These genetic defects result in a 50% reduction in corresponding enzyme activity with an estimated prevalence of 5% in the general population [70]. However, not all individuals with these deficiencies demonstrate elevated plasma homocysteine, suggesting other necessary conditions for full phenotypic expression [71, 72]. Genest et al. [59] evaluated plasma homocysteine in families of patents 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 [73]. 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 progression 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 targeted 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 greater in patients with elevated plasma homocysteine (Figure 4). Multiple regression analysis demonstrated that homocysteine correlated with progression independent of other established atherosclerotic risk factors. In a case-control study, elevated plasma homocysteine levels were associated with an increased likelihood of carotid artery intimal-medial wall thickness in symptomless adults [74]. A recent study examined homocysteine levels in blood samples obtained prospectively during the ongoing Physicians Health Study. Data from this study demonstrated a relative risk of 3.1 for myocarCARDIOVASCULAR SURGERY

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Figure 3 Presence of clinical and laboratory evidence of progression of disease in patients with normal and elevated plasma homocysteine. Clin, clinical progression; LED, lower extremity disease; CVD carotid artery disease; CAD, coronary artery disease. Percent refers to percentage of all patients in each category showing evidence of disease progression. (Used with permission from Taylor, L.M., Jr and Porter, J.M.,1993 [20].

Figure 4 Rate of clinical progression of disease (lower extremity plus carotid artery plus coronary artery disease) in patients with elevated plasma homocysteine compared with patients with normal plasma homocysteine. CPI, Clinical Progression Index (calculated by dividing the length of clinical follow-up in months by the number of clinical progression events). (Used with permission from Taylor, L.M., Jr and Porter, J.M., 1993 [20].)

dial infarction in men with homocysteine values at or above the 90th percentile [75]. This represents the first evidence identifying elevated plasma homocysteine as a significant risk factor for atherosclerosis obtained in a prospective manner (although analyzed retrospectively). A prospective study of plasma homocysteine and the progression of peripheral and cerebral vascular disease is ongoing [76].

Basic science data Homocysteine produces atherosclerotic vascular lesions when infused in experimental animals [11] Multiple research efforts have examined potential mechanisms for the atherogenicity of homocysteine. These studies have primarily focused on the poten563

Homocysteinemia: a risk factor for atherosclerosis: M. R. Nehler et al.

tial production of toxic by-products by the thiol side group of the homocysteine molecule. Several investigations of both 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 [77], but has shown that elevated plasma homocysteine acts in concert with dietary lipids [78]. He has suggested that homocysteine accumulation causes abnormal sulfuration of proteoglycans through the intermediary homocysteine thiolactone, a known cellular toxin [79, 80]. Using cultured endothelial cells from normal subjects, Wang et al. [81] noted measurable activity for only two of the three cellular enzymes involved in intracellular homocysteine metabolism. Activity level for methyltransferase was negligible. Due to this inherent limitation of the re-methylation pathway in the endothelium, they postulated these cells would be unable to process elevated plasma homocysteine if the transsulfuration pathway was impaired, as in the heterozygous state for cystathionine β-synthase deficiency. The resultant elevation in intracellular homocysteine would lead to cellular injury. In another experimental 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 [82]. This process was inhibited by catalase, suggesting that hydrogen peroxide was the oxidant agent produced. Another report using this model described endothelial cell lysis with homocysteine and copper exposure over time, again inhibited by catalase [83]. Despite this convincing animal data, in vivo studies have not demonstrated any difference in systemic peroxidation by-products in small numbers of patients with homocysteinemia compared with controls [84, 85]. Elevated homocysteine appears to impair the activity of several endothelial cell surface anticoagulants. In vitro models of homocysteinemia have demonstrated a reduction in the measured activity of protein C [86], possibly due to reduced activity of thrombomodulin, a known cofactor in protein C activation [87]. Alteration in endothelial cell surface proteins may be due to defects in intracellular transport secondary to oxidative changes caused by homocysteine [88]. In vitro studies have demonstrated excess homocysteine increases plateletderived thromboxane B2 and reduce endothelialderived prostacyclin [89]. Early structural changes in endothelium exposed to elevated homocysteine resemble those observed in atherosclerosis. In a pig model, homocysteinemia produces loss of elastic lamina and hypertrophy of muscle cells in the media. 564

These effects were partially eliminated with the use of a captopril/thiazide regimen [90]. 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, smoking cessation, hypertension control, and moderate exercise programs. These require major changes in life-styles and addictive behavior patterns, thus severely limiting overall success in large populations. Abundant information indicates that elevated plasma homocysteine can be reduced to normal with oral folate in most patients [91–93]. Individuals with homocysteine levels resistant to oral folate generally respond to non-toxic doses of pyridoxine, vitamin B12, choline or betaine [94, 95]. Interestingly, folate appears capable of normalizing homocysteine levels independent of the etiology, and appears effective regardless of the initial plasma folate levels. Vitamin B12 is somewhat less effective than folate in reducing plasma homocysteine [96], and pyridoxine appears useful only in cases with documented deficiency. Recently, several large trials have tested the ability of various vitamin regimens to reduce plasma homocysteine in large populations. Naurath et al. [97] in a prospective double-blinded, multicenter trial treated 285 unselected elderly persons (aged 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 with controls, irrespective of pre- or post-treatment vitamin levels. Two additional trials [98, 99] 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 ⱖ 95% percentile. All individuals identified as homocysteinemic were treated with 6 weeks of folate with pyridoxine added in some cases. Some 90–95% of those treated 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 [100]. Despite the enthusiasm of the medical community, the most important issue remains unanswered. Will normalization of elevated plasma homocysteine in patients CARDIOVASCULAR SURGERY

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with symptomatic atherosclerosis prove efficacious in preventing and/or arresting the disease process? The next decade will likely provide the answer to this question, and hopefully define the role of homocysteine reduction in the prevention/treatment of atherosclerosis.

18.

19.

20.

Acknowledgements These studies were supported by grant 1RO1HL45267-01A1, NIH, NHLBI, and grant MO1 RR00334, NIH, GCRCB

21.

22.

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