Prospective blinded study of the relationship between plasma homocysteine and progression of symptomatic peripheral arterial disease

Prospective blinded study of the relationship between plasma homocysteine and progression of symptomatic peripheral arterial disease Lloyd M. Taylor, Jr, MD, Gregory L. Moneta, MD, Gary J. Sexton, PhD, Robert A. Schuff, MS, John M. Porter, MD, and The Homocysteine and Progression of Atherosclerosis Study Investigators Portland, Ore Purpose: An elevated plasma homocysteine level is an established risk factor for atherosclerotic coronary heart disease (CHD), cerebrovascular disease (CVD), and lower extremity occlusive disease (LED). An elevated plasma homocysteine level can be reduced by therapy with folate and vitamins B6 and B12. An accurate evaluation of the role of vitamin therapy requires knowledge of the influence of plasma homocysteine levels on the progression of CHD, CVD, and LED. Methods: The Homocysteine and Progression of Atherosclerosis Study is a blinded prospective study of the influence of homocysteine and of other atherosclerotic risk factors on the progression of disease in patients with symptomatic CVD, LED, or both. This study is set in a university hospital vascular surgery clinic and the General Clinical Research Center. Consecutive patients with stable symptomatic CVD or LED underwent baseline clinical, laboratory, and vascular laboratory testing for homocysteine and other risk factors and were examined every 6 months. The primary endpoints were ankle brachial pressure index, duplex scan determined carotid stenosis, and death. The secondary endpoints were the clinical progressions of CHD, LED, and CVD. The hypothesis that was tested was whether the progression of symptomatic CVD or LED was more frequent or more rapid in patients with elevated plasma homocysteine levels. Results: After a mean follow-up period of 37 months (range, 1 to 78 months) for deaths from all causes (>14 µmol/l; elevated, 18.6%; normal, 9.4%; P =.022), deaths from cardiovascular disease (elevated, 12.5%; normal, 6.3%; P =.05) and the clinical progression of CHD (highest 20% of homocysteine levels, 80%; lowest 20% of homocysteine levels, 39%; P =.007) were significantly more frequent or more rapid by life-table analysis when the homocysteine levels were elevated. Multivariate Cox proportional hazards regression model showed a significant independent and increasing relationship between the plasma homocysteine levels and the time to death (relative risk for highest one third of homocysteine values, 1.6; 95% confidence interval [CI], 1.04 to 2.56; P =.029; and relative risk for highest one fifth of homocysteine values, 3.13; 95% CI, 1.69 to 6.64; P =.0001). After an adjustment for age, smoking, hypertension, diabetes, cholesterol, and the vascular laboratory progression of CVD or LED, each 1.0 µmol/l increase in the plasma homocysteine levels resulted in a 3.6% increase (95% CI, 0.0% to 6.6%; P =.06) in the risk of death (all causes) at 3 years and a 5.6% increase (95% CI, 2.2% to 8.5%; P =.003) in the risk of death from cardiovascular disease. Conclusion: We conclude that elevated plasma homocysteine levels are associated significantly with death, with death from cardiovascular disease, and with the progression of CHD in patients with symptomatic CVD or LED. These results strongly mandate clinical trials of homocysteine-lowering vitamin therapy in such patients. (J Vasc Surg 1999;29:8-21.) From the the Division of Vascular Surgery, and the General Clinical Research Center, Oregon Health Sciences University. Presented at the Fifty-second Annual Meeting of The Society for Vascular Surgery, San Diego, Calif, June 9 10, 1998. Supported in part by grant no. RR00234, Clinical Research Centers Branch, and NIH grant no. 2R01HL4526705, NIH, NHLBI. Reprint requests: Lloyd M. Taylor Jr, MD, Professor of Surgery, Division of Vascular Surgery, OP-11, Oregon Health Sciences University, 3181 S W Sam Jackson Park Rd, Portland, OR 97201. Copyright 1999 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter. 0741-5214/99/$8.00 + 0 24/6/94351 8

Volume 29, Number 1 Taylor et al 9 Elevated plasma homocysteine level has been established as an independent risk factor for the presence of atherosclerotic vascular disease, including coronary heart disease (CHD), 1 cerebrovascular disease (CVD), 2 and lower extremity occlusive disease (LED). 3 The interest in homocysteine as a risk factor has been markedly increased by the observation that elevated homocysteine levels can be normalized or substantially reduced in most patients with the administration of folate, pyridoxine, or cobalamin. 4 There presently is tremendous speculation that the treatment of elevated homocysteine levels with vitamin therapy may result in an improved prognosis for patients with atherosclerosis. Because significant atherosclerotic disease is infrequently detected before the onset of symptoms, risk factor reduction is intended in most patients to reduce the progression of the established disease and to prevent cardiovascular events. Understanding the role of atherosclerosis risk factor reduction requires accurate knowledge of the natural history of atherosclerotic disease progression. Patients with symptomatic peripheral arterial disease, including CVD and LED, are well suited for longitudinal studies because of the ability of repeated noninvasive testing to objectively show the progression of atherosclerotic lesions independent of the occurrence of symptoms. A preliminary retrospective analysis of the clinical course of the patients with peripheral arterial disease who were followed in our clinic suggested that the elevated homocysteine levels were associated with a more rapid disease progression, whether the progression was assessed by clinical symptoms or by noninvasive vascular laboratory techniques. 5 The Homocysteine and Progression of Atherosclerosis Study (HPAS; Appendix 1) is a 2-phase clinical research study that was instituted in 1991 to examine, in a prospective blinded fashion, the relationship between homocysteine levels, the progression of symptomatic peripheral arterial disease, and vitamin treatment. The first phase of the HPAS was a blinded 3-year study of the influence of homocysteine levels on the natural history of peripheral arterial disease. The results of this study form the basis for this report. METHODS Patient Selection The HPAS is being conducted at Oregon Health Sciences University (OHSU) with the facilities and the personnel of the Division of Vascular Surgery and the General Clinical Research Center of OHSU. Consecutive patients who were in long-term follow-up with symptomatic LED (claudication, limb-threatening ischemia) or CVD (transient cerebral ischemia, stroke) in the Vascular Surgery Clinic of OHSU were recruited for the study if they had vascular laboratory evidence of atherosclerotic disease at the symptomatic site (ankle brachial pressure index [ABI] <0.90 or carotid stenosis >16% diameter reduction). To be eligible for the study, the patients had to have at least 1 unoperated patent carotid artery or at least 1 unoperated leg the study was intended to examine the progression of atherosclerosis in unoperated vessels rather than postoperative recurrent disease. The patients with unstable disease (limb-threatening ischemia, symptomatic carotid disease) were not enrolled until treatment had returned them to a stable symptomatic status. The HPAS is fully approved by the Institutional Review Board of OHSU and by the Advisory Committee of the OHSU General Clinical Research Center. All the subjects gave informed consent. Study Design The HPAS is a 2-phase sequential study. The first phase was a 3-year natural history study that documented the relationship between homocysteine levels and progression of disease. After 3 years of follow-up, the subjects have been randomized to the second phase, which is a double-blinded treatment with folic acid (4 mg/day) or placebo, with the follow-up period continued for an additional 5 years. The hypothesis being tested by the natural history phase of the study reported here is whether the progression of peripheral arterial disease is more frequent or more rapid in patients with elevated plasma homocysteine levels than in patients with normal plasma homocysteine levels. Baseline Data The initial subject evaluation included a medical history, a physical examination, a laboratory evaluation (Appendix 2), and vascular laboratory testing of the lower extremities and carotid arteries. The lower extremity testing included segmental limb pressures and ankle pressure response to treadmill walking. 6 The carotid artery examination used duplex scanning with the University of Washington assignment of arteries to 1 of 6 categories of carotid stenosis (category A, 0% stenosis; category B, 1% to 16% stenosis; category C, 17% to 49% stenosis; category D, 50% to 79% stenosis; category D+, 80% to 99% stenosis; category E, occluded). 7 Homocysteine Testing The total plasma homocysteine levels (the sum of free and protein-bound homocysteine, homocystine,

10 Taylor et al January 1999 Table I. The Homocysteine and Progression of Atherosclerosis Study endpoints Primary endpoints ABI Carotid stenosis Death Secondary endpoints LED Worsening ischemia Amputation Revascularization surgery Angioplasty CVD Stroke Transient cerebral ischemia Carotid surgery CHD Myocardial infarction Coronary surgery Coronary angioplasty Congestive heart failure New onset of angina Arrhythmia ABI, Ankle brachial systolic pressure ratio; LED, lower extremity occlusive disease; CVD, cerebrovascular disease; CHD, coronary heart disease. and the mixed disulfide homocysteine-cysteine) were measured with high performance liquid chromatography with fluorescence detection by a method published previously. 8 The samples for homocysteine testing were performed at the time of the study clinic visits without regard to fasting and without loading tests. The homocysteine level testing was performed at the time of study enrollment and at 6 monthly intervals. The initial homocysteine levels obtained in each subject at the time of study enrollment were used in all data analyses of the association between homocysteine levels and progression of disease. Follow-up Period All the patients were seen by one of the investigators every 6 months. An interval history and a repeat physical examination were performed, and the vascular laboratory testing was repeated. The plasma homocysteine testing also was repeated. Treatment The patients underwent standardized management of peripheral arterial disease directed by the investigators. The nonoperative management included daily aspirin (325 mg/day), counseling regarding smoking cessation, and a recommendation for daily walking exercise at every visit. The patients with lipid abnormalities were treated by the OHSU lipid and metabolic disorders clinic. Operations for lower extremity ischemia were recommended for disabling claudication or for limb-threatening ischemia and were performed by the investigators according to the principals published previously. 9 Carotid endarterectomy was recommended for the progression to symptomatic stenosis >70% diameter reduction for patients who met other published eligibility criteria for the NASCET study 10 or for the progression of asymptomatic stenosis to >60% diameter reduction for patients who met other published criteria for entry into the ACAS study. 11 Progression of Disease The primary endpoints for the study were ABI, degree of carotid stenosis, and death. Death from all causes was chosen as the primary endpoint to eliminate any confounding source of error regarding causes of death, but death from cardiovascular causes also was analyzed. The secondary endpoints included the clinical progression of CHD, LED, and CVD (Table I). Any of the secondary endpoints listed in Table I were regarded as evidence of the clinical progression of disease. The progression of LED was defined as an ABI decrease of more than 0.15. The progression of carotid stenosis was defined as an increase in at least 1 category of stenosis. The cause of death was determined from the hospital discharge summaries or the death certificates. The nonfatal clinical progression of CHD, LED, and CVD was determined by hospital discharge diagnoses, by patient interview at the regularly scheduled follow-up visits, and by communication with the referring physicians. Data Monitoring Committee All the study data were examined by a data monitoring committee. For the natural history portion of the study, the responsibility of the data monitoring committee was to identify if and when a significant difference in primary endpoints related to plasma homocysteine levels existed. If this difference occurred, the committee was instructed to unblind the group results of the natural history portion of the study so that all the patients could be randomized to the treatment trial. During the treatment trial, the responsibility of the data monitoring committee is to identify if and when a significant difference in primary endpoints existed so that the trial could be stopped. Blinding For the natural history phase of the HPAS all the study data were kept in a dedicated database, with patients identified by number only. The investigators were blinded to the patient homocysteine levels. The technicians who performed the homocysteine level

Volume 29, Number 1 Taylor et al 11 Table II. Selected baseline laboratory data (mean ± standard deviation of the mean) Parameter Elevated homocysteine levels (n = 131) Normal homocysteine levels (n = 220) P* Cholesterol 227 ± 53 mg/dl 215 ± 42 mg/dl.04 Cotinine 216 ± 261 ng/ml 159 ± 251 ng/ml.05 Creatinine 1.39 ± 0.94 mg/dl 1.06 ± 0.23 mg/dl.002 Folate 11.06 ± 14.8 ng/ml 15.82 ± 9.93 ng/ml.002 B12 402.5 ± 185.1 pg/ml 549.2 ± 482.5 pg/ml.0001 B6 20.81 ± 30.44 nmol/l 50.04 ± 89.24 nmol/l.0001 Viscosity (cp) 1.68 ± 0.096 cp 1.67 ± 0.170 cp.28 Leukocytes 8329 ± 3000 mm 3 7880 ± 2284 mm 3.15 Elevated, Plasma homocysteine level >14 µmol/l; normal, plasma homocysteine level <14 µmol/l. *Probability with Student t test. Smoking was assessed with plasma cotinine levels. tests were blinded to patient identity. The vascular laboratory technicians were blinded to the patient homocysteine levels and to the results of the previous vascular laboratory testing. Blinding of the study with respect to data analysis was maintained by presenting the data to the data monitoring committee with the patient groups identified only as group A or group B, etc, with no indication as to which group had the normal homocysteine levels and which had the elevated levels. Only when a significant difference between the groups was clearly identified by the data monitoring committee were the identities of the groups revealed. Data Analysis The HPAS data were recorded on study worksheets and entered into a microcomputer database on a weekly basis. Yearly analyses of the primary and secondary endpoint outcomes was performed in a blinded fashion. The baseline laboratory and clinical data were compared with Student t test for continuous variables and χ 2 test for categorical variables. The primary and secondary outcomes were evaluated with the life-table analysis method. The life tables have been compared with the log-rank test. Multivariate analysis was performed with the Cox proportional hazards model for censored data, with time to the various primary and secondary endpoints as the dependent variable. The model originally was constituted with all the relevant study data as independent variables and then, in sequential fashion, refining the model to include only those independent variables that had a significant influence on the time to the various endpoints. All multivariate analyses of the vascular laboratory progressions were corrected for the influence of the number of arteries available for assessment because various subjects had the potential for detection of progression in 1 to 4 sites. The influence of the plasma homocysteine levels on time to endpoint occurrence was evaluated from the standpoint of the elevated versus the normal levels in a stepwise fashion (eg, the highest 25% vs the lowest 25%) and as a continuous variable. RESULTS Blinding The blinded annual data analysis that was performed in November 1997 showed a significant difference in the deaths from any cause and the deaths from cardiovascular causes related to plasma homocysteine levels. On the basis of this analysis, the data monitoring committee (Appendix 1), as specified in the protocol, recommended unblinding the results of the natural history portion of the HPAS. Only the group results as reported in this manuscript were unblinded. The investigators remained blinded to the individual subject plasma homocysteine levels so that the results of the folic acid treatment trial portion of the HPAS remained blinded and the data continue to be presented to the data monitoring committee in a blinded fashion. The results reported in this paper are those of a subsequent unblinded analysis of the natural history portion of the HPAS that was performed in April 1998. Subjects Of the 408 subjects who consented to be recruited into the study, 33 (8%) did not complete the baseline testing and were regarded as not having entered the study. Of the 375 subjects who completed the baseline testing, 24 (6%) did not return for follow-up visits or withdrew from the study. The demographic and clinical data for the 57 subjects who were initially recruited but who did not return or withdrew from the study and the laboratory data including the homocysteine levels for the 24 who completed the baseline testing but did not return or withdrew were not different when compared with

12 Taylor et al January 1999 Table III. Selected baseline demographic characteristics of subjects Parameter Elevated homocysteine levels (n = 131) Normal homocysteine levels (n = 220) P* Age (mean ± SD; years) 66.31 ± 9.03 65.89 ± 8.95.67 Male gender 92 (70%) 130 (59%).04 CHD 72 (55%) 101 (46%).10 CVD 42 (32%) 82 (37%).32 LED 118 (90%) 178 (81%).03 Diabetes 24 (18%) 57 (26%).10 HTN 87 (66%) 123 (56%).05 Elevated, Plasma homocysteine level >14 µmol/l; normal, plasma homocysteine level <14 µmol/l; SD, standard deviation of the mean; CHD, coronary heart disease; CVD, cerebrovascular disease; LED, lower extremity ischemia; HTN, hypertension. *Probability with Student t test (age) or with χ 2 test. that of the 351 subjects who remained in the study (P = ns for all comparisons; data not shown). The 351 subjects, whose mean age was 66 years (range, 40 to 80 years), included 222 males (63%). LED symptoms were present in 296 subjects (85%), CVD symptoms were present in 124 subjects (35%), and 86 subjects (25%) had both LED and CVD symptoms. Other selected baseline characteristics of the 351 subjects are listed in Tables II and III. The subjects with elevated plasma homocysteine levels were significantly more likely to be men (P =.04) and to have LED (P =.03) and hypertension (P =.05). The subjects with elevated plasma homocysteine levels also had significantly higher levels of cholesterol (P =.04), cotinine (P =.05), and creatinine (P =.002) and significantly lower levels of folate (P =.002), B12 (P =.001), and B6 (P =.0001). There were no significant differences in the numerous other laboratory parameters as listed in Appendix 2, including viscosity, leukocyte count, serum protein levels, presence of hypercoagulable states, and lipoprotein (a) levels (data not shown). Follow-up Period The mean follow-up period for all the subjects was 37 months (range, 1 to 78 months; standard deviation of the mean, 20.13 months). The followup period for the subjects with elevated homocysteine levels was significantly shorter than that for the subjects with normal homocysteine levels (33.1 ± 19.6 months vs 40.5 ± 19.9 months; P =.0008, with Student t test). The difference in follow-up period is explained by the more frequent occurrence of early death in subjects with elevated homocysteine levels. Plasma Homocysteine Levels The mean plasma homocysteine level for the 351 subjects at the time of study entry was 14.029 µmol/l (range, 5.6 to 64.1 µmol/l; standard deviation of the mean, 6.927 µmol/l). The plasma homocysteine testing on the volunteer controls without symptoms or vascular laboratory evidence of atherosclerotic disease in the atherosclerotic age group (n = 38; 20 men; mean age, 64 years) gave a mean value of 10.2 ± 1.9 µmol/l. On the basis of this testing, the HPAS subjects with initial plasma homocysteine values of more than 14.0 µmol/l (2 standard deviations above the control mean value) were regarded as having elevated plasma homocysteine levels and those with values of less than 14.0 µmol/l were regarded as having normal levels. The plasma homocysteine values were repeated for all the subjects every 6 months. There were no significant changes in the study population homocysteine level values for mean, median, range, or standard deviation related to date or to length of follow-up period (data not shown). The plasma homocysteine level was inversely related to folate (R = 0.234, P =.001), B6 (R = 0.157, P =.004), and B12 (R = 0.153, P =.005) and was directly related to creatinine (R = 0.235, P =.001) as was expected. There were no other significant relationships between plasma homocysteine levels and any of the parameters listed in Appendix 2 (data not shown). Progression of Disease, Univariate Analysis Death. Forty-seven deaths (13%) occurred during the first 3 years of the follow-up period at intervals that ranged from 1 to 36 months 33 of these deaths (70%) were caused by cardiovascular disease. According to life-table analysis (Fig 1), after 3 years of follow-up, the deaths from any cause and the deaths from cardiovascular disease were significantly more likely in the subjects with elevated plasma homocysteine levels (death from any cause, 9.4% normal homocysteine level vs 18.6% elevated homocysteine level; P =.02; death from cardiovascular disease, 6.3% normal homocysteine level vs 12.5% ele-

Volume 29, Number 1 Taylor et al 13 A B Fig 1. Life-table analysis of survival rates from death from any cause (A) and death from cardiovascular disease (B) that compares subjects with elevated plasma homocysteine levels (>14.0 µmol/l) with those with normal plasma homocysteine levels (<14.0 µmol/l). vated homocysteine level; P =.05). Both the absolute magnitude of the differences in death and survival rates and the significance of the differences increased progressively with the magnitude of the differences in plasma homocysteine levels. For example, the subjects with the highest 33% of plasma homocysteine values had an 80% survival rate with life-table analysis after 3 years, but those with the lowest 33% of plasma homocysteine values had a 93% survival rate (P =.017, with log-rank test). In contrast, the subjects with the highest 20% of plasma homocysteine values had a 71% survival rate by lifetable analysis after 3 years, but those with the lowest 20% of plasma homocysteine values had a 95% survival rate (P =.0006, with log-rank test; Table IV). Nonfatal clinical progression. The subjects with the highest levels of homocysteine were significantly more likely to have nonfatal clinical progression of CHD (upper 20%, 80% progression with lifetable analysis after 3 years; lower 20%, 39% progression after 3 years; P =.0068, with log-rank test). The clinical progression of LED was more frequent in the subjects with the highest homocysteine levels (upper 20%, 15% progression; lower 20%, 7.3% progression), but the difference was not significant (P =.310, with log-rank test). There was no difference in the clinical

14 Taylor et al January 1999 Table IV. Life-table analysis survival rates after 3 years Groups compared n Survival rates (%) P* All elevated homocysteine levels 131 81.023 All normal homocysteine levels 220 91 Highest 33% homocysteine levels 120 80.017 Lowest 33% homocysteine levels 109 93 Highest 25% homocysteine levels 92 77.005 Lowest 25% homocysteine levels 83 94 Highest 20% homocysteine levels 72 71.0006 Lowest 20% homocysteine levels 69 95 Elevated, Plasma homocysteine level >14 µmol/l; normal, plasma homocysteine level <14 µmol/l; highest 33%, 1/3 of subjects with highest plasma homocysteine values (14.9 to 64.1 µmol/l); lowest 33%, 1/3 of subjects with lowest plasma homocysteine values (5.6 to 10.6 µmol/l); highest 25%, 1/4 of subjects with highest plasma homocysteine values (16.7 to 64.1 µmol/l); lowest 25%, 1/4 of subjects with lowest plasma homocysteine values (5.6 to 9.8 µmol/l); highest 20%, 1/5 of subjects with highest plasma homocysteine values (17.9 to 64.1 µmol/l); lowest 20%, 1/5 of subjects with lowest plasma homocysteine values (5.6 to 9.3 µmol/l). *Probability with log-rank test. Table V. Nonfatal clinical progression of atherosclerosis Parameter Group Percentage with progression after 3 years* (%) P CHD Highest 20% homocysteine level 80.0068 Lowest 20% homocysteine level 39 CVD Highest 20% homocysteine level 11.659 Lowest 20% homocysteine level 9 LED Highest 20% homocysteine level 15.31 Lowest 20% homocysteine level 7 Stroke Highest 20% homocysteine level 9.279 Lowest 20% homocysteine level 2 MI Highest 20% homocysteine level 11.454 Lowest 20% homocysteine level 5 Worse ischemia Highest 20% homocysteine level 15.171 Lowest 20% homocysteine level 3 CHD, Coronary heart disease; CVD, cerebrovascular disease; LED, lower extremity disease; MI, myocardial infarction; worse ischemia, increase in severity of lower extremity ischemia; highest 20% homocysteine level, 1/5 of subjects with highest homocysteine levels (17.9 to 64.1 µmol/l), n = 72; lowest 20% homocysteine level, 1/5 of subjects with lowest homocysteine levels (5.6 to 9.3 µmol/l), n = 69. *With life-table analysis method. Probability with log-rank test. progression of CVD as related to the homocysteine levels (upper 20%, 9.5% progression; lower 20%, 10.5% progression; P = ns). After 3 years, all the clinical events, including stroke, limb-threatening ischemia, amputation, myocardial infarction, heart failure, arrhythmia, and need for carotid, coronary, or lower extremity revascularization surgery, were more frequent in the subjects with the highest homocysteine levels, but, except for CHD, none of the differences were statistically significant (Table V). Vascular laboratory progression. The progression of LED as assessed by decreasing ABI occurred more frequently in the subjects with the highest homocysteine levels (highest 20%, 33% progression with life-table analysis after 3 years; lowest 20%, 21%), but the difference was not significant (P =.20, with log-rank test). There was no difference in the progression of CVD as assessed by increasing carotid stenosis (highest 20%, 31% with life-table analysis after 3 years; lowest 20%, 29%; P =.79, with log-rank test). Progression of Disease, Multivariate Analysis Because of the significant differences between the patients with elevated homocysteine levels and those with normal homocysteine levels in gender, cholesterol, smoking (as reflected by cotinine levels), creatinine, vitamin status, and hypertension, the influence of the plasma homocysteine levels on the progression of disease was examined extensively with multivariate analysis. As was expected, the levels of folate, B6, and B12 were correlated with death whenever there was an association with the homocysteine levels, but in no case was the association stronger than in that found with homocysteine levels. For this reason, the vitamin levels were eliminated from further consideration in the multivariate model.

Volume 29, Number 1 Taylor et al 15 Table VI. Relative risk of death* associated with various atherosclerosis risk factors Factor Relative risk of death 95% CI P Elevated homocysteine level 1.30 0.96 to 1.46.088 Hypertension 1.47 1.05 to 2.12.024 Age 1.03 1.00 to 1.07.049 Smoking 1.001 1.00 to 1.002.043 VL progression 1.58 1.17 to 2.19.003 CI, Confidence interval; elevated homocysteine level, plasma homocysteine >14 µmol/l; age, relative risk as a result of increasing age in yearly increments more than age 40 years; smoking, relative risk as a result of each mg/ml increase in plasma cotinine more than 150 mg/ml; VL progression, relative risk as a result of occurrence of progression of lower extremity or carotid artery disease by vascular laboratory testing. *Influence of elevated homocysteine levels and other risk factors found to significantly influence time to death in a Cox proportional hazards model that originally considered age, gender, diabetes, cholesterol, smoking, hypertension, CHD, LED, CVD, vascular laboratory progression of CVD or LED, creatinine, levels of folate, B12, B6, and plasma homocysteine. Insignificant variables were eliminated in sequential fashion to determine the relative risk of those remaining significant. Probability with likelihood ratio testing. Table VII. Relative risk of death* associated with increasing levels of plasma homocysteine Homocysteine group Relative risk 95% CI P Elevated homocysteine level vs normal homocysteine level 1.30 0.96 to 1.46.088 Highest 33% homocysteine level vs lowest 33% homocysteine level 1.61 1.04 to 2.56.029 Highest 25% homocysteine level vs lowest 25% homocysteine level 2.02 1.23 to 3.51.005 Highest 20% homocysteine level vs lowest 20% homocysteine level 3.13 1.69 to 6.64.0001 CI, Confidence interval; highest 33% homocysteine level, 1/3 of subjects with highest homocysteine levels (14.9 to 64.1 µmol/l); lowest 33% homocysteine level, 1/3 of subjects with lowest homocysteine levels (5.6 to 10.6 µmol/l); highest 25% homocysteine level, 1/4 of subjects with highest homocysteine levels (16.7 to 64.1 µmol/l); lowest 25% homocysteine level, 1/4 of subjects with lowest homocysteine levels (5.6 to 9.8 µmol/l); highest 20% homocysteine level, 1/5 of subjects with highest homocysteine levels (17.9 to 64.1 µmol/l); lowest 20% homocysteine level, 1/5 of subjects with lowest homocysteine levels (5.6 to 9.3 µmol/l). *Influence of plasma homocysteine levels on time to death in a Cox proportional hazards model that included age, smoking, hypertension, and vascular laboratory progression of disease. Probability with likelihood ratio testing. Death, multivariate analysis. Table VI shows the relative risk of death associated with the various risk factors found to be significant in the multivariate model, which originally included age, gender, diabetes, cholesterol, smoking, hypertension, CHD, CVD, LED, vascular laboratory progression of CVD or LED, creatinine, levels of folate, B12, B6, and plasma homocysteine. An elevated plasma homocysteine level (>14 µmol/l) had a moderate independent influence on death (relative risk, 1.24; 95% confidence interval [CI], 0.96 to 1.46; P =.088) that was not significant by the criterion of P less than.05 and was of lower magnitude than other significant factors (Table VI). The progression of CVD or LED documented in the vascular laboratory showed a highly significant independent association with death (relative risk 1.58; 95% CI, 1.17 to 2.19; P =.003). Table VII shows the progressively increasing independent influence of plasma homocysteine on time to death as the level of homocysteine increased. For example, the relative risk of death with a plasma homocysteine level of more than 14 µmol/l as compared with less than 14 µmol/l was 1.31 (95% CI, 0.96 to 1.77; P =.09), and this increased to a relative risk of 3.13 (95% CI, 1.68 to 6.64; P =.001) when the subjects with the highest 20% of homocysteine levels were compared with those with the lowest 20% of homocysteine levels (Table VII). The vascular laboratory progression of CVD or LED remained significantly independently associated with death as increasing levels of homocysteine were examined in the multivariate model. After adjustment for age, hypertension, smoking, and vascular laboratory progression of CVD or LED, each increase of 1 µmol/l of plasma homocysteine level resulted in a 3.6% increase in the risk of death (95% CI, 0.0% to 6.6%; P =.06), although this relationship was not significant by the criterion of P less than.05. The relationship was more pronounced when the analysis was confined to death from cardiovascular disease. After adjustment for age, hypertension, smoking, diabetes, and cholesterol, each increase of 1 µmol/l of plasma homocysteine level resulted in a 5.6% increase in the like-

16 Taylor et al January 1999 A B Fig 2. Increasing relative risk of death (A) and of death from cardiovascular disease (B) associated with increasing levels of plasma homocysteine (in µmol/l). Brackets indicate 95% confidence interval. lihood of death from vascular disease (95% CI, 2.2% to 8.5%; P =.003). Fig 2 shows a graphic representation of the increasing independent risk of death and death from vascular disease associated with increasing levels of plasma homocysteine. DISCUSSION Since the initial description by Wilcken and Wilcken 12 in 1976, a large number of studies have confirmed that an elevated plasma homocysteine level is an independent risk factor for the presence of atherosclerotic cardiovascular disease. The relationship has been confirmed for patients with CVD, LED, and CAD, regardless of gender or age and regardless of whether the homocysteine levels were assessed randomly, by fasting, or after methionine loading. 13 An elevated homocysteine level is important as a risk factor not only because of the strength of its association with the presence of atherosclerotic disease but also because of the relationship of the plasma levels of this metabolic intermediary to the levels of the vitamins folate, B6, and B12. In humans, homocysteine is formed primarily from dietary methionine and is metabolized either to cysteine via the transulfuration pathway for which B6 is a cofactor or back to methionine via remethylation for which the primary methyl

Volume 29, Number 1 Taylor et al 17 donor is tetrahydrofolate and for which B12 is a cofactor. Numerous studies have shown that the levels of plasma homocysteine are inversely related to the levels of folate, B6, and B12, 14-16 a relationship that is confirmed in this study. The ability to therapeutically reduce elevated levels of homocysteine with the administration of folate and, to a lesser degree, B6 and B12 has also been repeatedly shown. 17-19 The obvious question is whether vitamin treatment might have benefit in the prevention or treatment of atherosclerotic cardiovascular disease. Although some investigators have recommended the treatment of elevated homocysteine levels on the basis of current knowledge, in our opinion, this approach is not justified. There are obvious differences between association and causation, and not all associations or causations can be modified with therapy. The unanticipated negative effect that is associated with the widely heralded treatment with antioxidants and beta carotene is an excellent example. 20 The proper scientific approach is to conduct appropriate clinical trials, as has been suggested by other investigators. 21 An evaluation of the potential effects of vitamin treatment on atherosclerotic disease requires the knowledge of the precise effect of homocysteine levels on the natural history of disease. To date, only a few studies have evaluated the relationship between homocysteine levels and atherosclerotic disease prospectively. Stampfer and colleagues 22 examined prospectively obtained blood samples from the Physicians Health Study, and the risk of myocardial infarction was 3.1 times higher in men with plasma homocysteine values in the highest 5%. Alfthan and colleagues 23 found no relationship between homocysteine levels and atherosclerotic disease in a Finnish population. Arneson and coauthors 24 showed that elevated homocysteine levels predicted myocardial infarction in a general population in Norway. Perry and coauthors 25 and Verhoef and colleagues 26 both found that elevated homocysteine levels were related to ischemic strokes. Recently, Norwegian investigators prospectively demonstrated a strong relationship between homocysteine levels and early mortality rates in patients with established CHD. 27 To our knowledge, the HPAS is the first prospective blinded study to show a significant independent relationship between plasma homocysteine levels and mortality rates in patients with symptomatic atherosclerotic disease. The relative risk of death found in the present study is similar to those documented in the other available prospective studies (Tables VI, VII). 22,24,27 Although this phase of the HPAS clearly has documented a relationship between increasing plasma homocysteine levels and increasing risk of early death from cardiovascular disease, there was no similar relationship with the progression of disease as documented in the noninvasive vascular laboratory. Decreasing ABI and increasing carotid stenosis both were more frequent in the subjects with elevated homocysteine levels, but the difference was not significant. This may be because the study was too short in duration or too small, or it may be because the patient population with elevated homocysteine levels was rapidly censored by early death, which prevented detection of a difference in the vascular laboratory progression. This hypothesis is supported by the strong independent association between the vascular laboratory progression and the time to death (P =.003; Table VI). Alternately, the elevated plasma homocysteine may be more strongly related to the thrombotic complications of atherosclerosis than to the actual progression of atherosclerotic lesions, as suggested by the multiple laboratory studies that showed an influence of homocysteine at multiple points in the thrombotic process. 28-30 In the present study, this possibility is supported by a stronger association between the elevated homocysteine levels and the ABI progression (P =.20) than the carotid stenosis progression (P =.79) because the changes in ABI reflect the combined influence of increasing atherosclerosis and thrombosis whereas the increasing carotid stenosis is related almost entirely to the increasing amounts of atherosclerotic plaque. At present, this must be regarded as speculation. Against this possible mechanism are studies that show a direct relationship between the plasma homocysteine levels and the increasing atherosclerotic plaque, as assessed with coronary arteriography, 31 carotid artery wall thickness, 32 and carotid artery stenosis. 33 It is possible that the 24 subjects (6%) who were initially recruited into the study but were lost to follow-up might have influenced the result, if they had remained. The lack of difference in all clinical and laboratory parameters between these subjects and the 351 (94%) who remained in the study makes this possibility extremely unlikely. Multiple factors are associated with the progression of atherosclerotic disease. In the present study, the subjects with elevated plasma homocysteine levels also had significantly higher cholesterol levels (P =.04; Table II), smoking levels, as assessed with cotinine levels (P =.05; Table I), and hypertension levels (P =.05; Table III), each of which was shown

18 Taylor et al January 1999 to have a significant independent relationship to time to death in the multivariate analysis (Table VI). More subjects with elevated plasma homocysteine levels also had LED, which has been associated with early death from cardiovascular disease. 34 The vascular laboratory progression of disease was confirmed as an independent risk factor for time to death in the multivariate model (P =.003; Table VI). Despite these differences and associations, the plasma homocysteine levels were independently associated with death (P =.088) and death from cardiovascular disease (P =.003), and the strength of the association increased markedly with the increasing homocysteine levels even when adjusted for the other significant independent associations (Table VII). The magnitude of the association between the increasing plasma homocysteine levels and the increasing cardiovascular mortality rates (Fig 2) is similar to the magnitude of the association between the increasing levels of cholesterol and cardiovascular mortality rates, as noted by other investigators. 35 We conclude that the HPAS has shown, in prospective blinded fashion, that elevated plasma homocysteine levels are independently associated with death, death from cardiovascular disease, and nonfatal progression of CHD in patients with symptomatic peripheral arterial disease. The relationship is graded with increasing levels of plasma homocysteine and is similar in importance to that associated with other common risk factors, such as hypertension, cholesterol, smoking, and age. These results significantly add to the mandate for clinical trials to examine the effect of homocysteine-lowering vitamin therapy on cardiovascular morbidity and mortality rates. REFERENCES 1. Moghadasian MH, McManus BM, Frohlich JJ. Homocyst(e)ine and coronary artery disease. Arch Intern Med 1997;157:2299-308. 2. Coull BM, Malinow MR, Beamer N, Sexton G, Nordt F, de Garmo P. Elevated plasma homocyst(e)ine concentration as a possible independent risk factor for stroke. Stroke 1990; 21:572-6. 3. Malinow MR, Kang SS, Taylor LM Jr, Wong PWK, Coull B, Inahara T, et al. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation 1989;79:1180-8. 4. Brattstrom L. Vitamins as homocysteine-lowering agents. J Nutr 1996;126:S1276-80. 5. 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 Vasc Surg 1991;13:128-36. 6. Baur GM, Zupan TL, Holmgren-Gates K, Porter JM. Blood flow in the common femoral artery. Am J Surg 1983;145: 585-8. 7. Strandness DE Jr. Extracranial arterial disease. In: Strandness DE Jr, editor. Duplex scanning in vascular disorders. 2nd ed. New York: Raven Press; 1993. p. 113-58. 8. Jacobsen DW, Gateutis VJ, Green R, Robinson K, Ji J, Savon SR, 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 Chem 1994;40:873-81. 9. Yeager RA, Taylor LM Jr, Porter JM. The present status of infrainguinal arterial reconstructive surgery for chronic lower extremity ischemia. Curr Probl Surg 1991;28:125-39. 10. North American Symptomatic Carotid Endarterectomy (NASCET) Steering Committee. North American Symptomatic Carotid Endarterectomy Trial: methods, patient characteristics, and progress. Stroke 1991;22:711-20. 11. Asymptomatic Carotid Atherosclerosis Study Group. Study design for randomized prospective trial of carotid endarterectomy for asymptomatic atherosclerosis. Stroke 1989;20:844-9. 12. Wilcken DEL, Wilcken B. The pathogenesis of coronary artery disease: a possible role for methionine metabolism. J Clin Invest 1976;57:1079-82. 13. Nehler MR, Taylor LM Jr. Homocysteinemia as a risk factor for atherosclerosis: a review. Cardiovasc Surg 1997;5:559-67. 14. Jacob RA, Wu MM, Henning SM, Swendseid ME. Homocysteine increases as folate decreases in plasma of healthy men during short-term dietary folate and methyl group restriction. J Nutr 1994;124:1072-80. 15. Miller JW, Ribaya-Mercado JD, Russell RM, Shepard DC, Morrow FD, Cochary EF, et al. Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr 1992;55:1154-60. 16. Brattstrom L, Israelsson B, Lindgarde F, Hultberg B. Higher plasma homocysteine in vitamin B12 deficiency than in heterozygosity for homocystinuria due to cystathionine B-synthase deficiency. Metabolism 1988;37:175-8. 17. Olszewski AJ, Szostak WB, Bialkowska M, et al. Reduction of plasma lipid and homocysteine levels by pyridoxine, folate, choline, riboflavin, and troxerutin in atherosclerosis. Atherosclerosis 1989;75:1-6. 18. Wilcken DEL, Dudmon NPB, Tyrell P, Robertson MR. Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency possible implication for prevention of vascular disease. Metabolism 1988;37:697-701. 19. Brattstrom L. Vitamins as homocysteine-lowering agents. J Nutr 1996;126:S1276-80. 20. Omenn GS, Goodman GE, Thornquist MD, et al. Effects of a combination of beta-carotene and vitamin A on lung cancer incidence, total mortality, and cardiovascular mortality in smokers and asbestos-exposed workers. N Engl J Med 1996;334:1150-5. 21. Malinow MR. Hyperhomocyst(e)inemia. Circulation 1990; 81:2004-6. 22. 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. 23. Alfthan G, Pekkanen J, Jauhiainen M, et al. Relation of serum homocysteine and lipoprotein (a) concentrations to atherosclerotic disease in a prospective Finnish population study. Atherosclerosis 1994;106:9-19. 24. Arnesen E, Refsum H, Bonaa KH, et al. Serum total homocysteine and coronary heart disease. Int J Epidemiol 1995; 24:704-9. 25. Perry IJ, Refsum H, Morris RW, et al. Prospective study of

Volume 29, Number 1 Taylor et al 19 serum homocysteine concentration and risk of stroke in middle aged men. Lancet 1995;346:1395-8. 26. Verhoef P, Hennekens CH, Malinow MR, et al. A prospective study of plasma homocyst(e)ine and risk of ischemic stroke. Stroke 1994;25:1924-30. 27. Nygard O, Nordrehaug JE, Refsum H, et al. Plasma homocysteine levels and mortality in patients with coronary heart disease. N Engl J Med 1997;337:230-6. 28. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood 1990;75:895-901. 29. 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. 30. Harpel PC, Chang VT, Borth W. Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein (a) to fibrin: a potential biochemical link between thrombosis, atherogenesis, and sulfhydryl compound metabolism. Proc Natl Acad Sci U S A 1992;89:10193-7. 31. Robinson K, Mayer EL, Miller DP, et al. Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825-30. 32. Malinow MR, Nieto FJ, Szklo M, Chambless LE, Bond G. Carotid artery intimal-medial thickening and plasma homocyst(e)ine in asymptomatic adults: the atherosclerosis risk in communities study. Circulation 1993;87:1107-13. 33. Selhub J, Jacques PF, Bostom AG, et al. Association between plasma homocysteine concentrations and extracranial carotidartery stenosis. N Engl J Med 1995;332:286-91. 34. McDermott MM, Feinglass J, Slavensky R, Pearce WH. The ankle-brachial index as a predictor of survival in patients with peripheral vascular disease. J Gen Intern Med 1994;9:445-9. 35. Graham IM, Daly LE, Refsum HM, et al. Plasma homocysteine as a risk factor for vascular disease: the European concerted action project. JAMA 1997;277:1775-81. Submitted Jun 11, 1998; accepted Sep 3, 1998. DISCUSSION Dr Hisham S. Bassiouny (Chicago, Ill). To date, research for a humoral marker that would reliably predict human atherosclerotic plaque progression and disruption has yielded inconclusive results. Nonetheless, plasma homocysteine, a metabolite of the amino acid methionine, deserves particular consideration because it is elevated in approximately 30% of patients with premature coronary and peripheral atherosclerosis. Another attractive feature about this potential risk factor is that elevated levels can be readily normalized with inexpensive folic acid and vitamin B6 and B12 supplementation. In 1969, McCully made the seminal observation that linked elevated plasma homocysteine levels with vascular disease in 2 children with autopsy evidence of extensive atherosclerosis and arterial thrombosis. Since, epidemiologic evidence amassed from large prospective trials, case control, and cross-sectional studies have validated this association. Hyperhomocysteinemia is now recognized as an independent risk factor for cardiovascular disease and events. For example, in the Physicians Health Study, which comprised 15,000 male subjects followed for 5 years, homocysteine levels 10% above the upper limit of normal range were accompanied with a 3-fold risk of myocardial infarction. A graded rather than a threshold relation between plasma homocysteine levels and carotid stenosis was additionally found in the Framingham study. Dr Taylor and his associates are to be congratulated on conducting a rigorous, well-controlled, and properly analyzed prospective study designed to elucidate the role of plasma homocysteine in 351 patients with noninvasive evidence of carotid and peripheral occlusive disease. Their multivariate analysis emphasizes the association of hyperhomocysteinemia with only 2 of the clinical endpoints selected more specifically, time to death from cardiovascular events and the clinical evidence of nonfatal progression of coronary heart disease. This relationship also appeared incremental such that increase of 1 µmol/l of plasma homocysteine was associated with approximately a 5.6% increase in the risk of cardiovascular death. However, the hypothesis being principally tested in the study was proven null. More specifically, no statistically significant increase in the relative risk of progression of either carotid or peripheral atherosclerosis was found in relation to plasma homocysteine levels. This may be attributed to a number of inherent study limitations, as Dr Taylor correctly points out in the manuscript, which include a short follow-up duration and, more importantly, the multitude of confounding atherosclerosis risk factors relative to the number of enrolled patients. In 1990, Dr Taylor presented data from a retrospective case control study of 317 patients regarding the role of plasma homocysteine in peripheral arterial disease and concluded that plasma homocysteine is an independent risk factor for progression of lower extremity but not carotid occlusive disease. This previously published positive association was not reproduced in this study. Perhaps you can provide us with an explanation for these equivocal results. My second question relates to the lack of correlation between increased risk of cardiovascular death and concomitant progression of plaque burden in the lower extremity and carotid vessels. This suggests that hyperhomocysteinemia may play a role in modifying plaque structural composition and its predisposition to complications, such as surface thrombosis and fibrous cap disruption. Have you found any differences between plaque structural