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1 From the University of Texas Southwestern Medical Center, Dallas, TX (BA-H, TS, AH, and JAdeL); University of California Davis Medical Center, Sacramento, CA (SD and IJ); and Wake Forest University, Winston-Salem, NC (RT)
2 Supported by grants from NIH (K24 AT 00596 and RO1 AT00005). 3 Address reprint requests to I Jialal, Laboratory for Atherosclerosis and Metabolic Research, University of California, Davis, Medical Center, 4635, Second Avenue, Research Building 1, Room 3000, Sacramento, CA 95817. E-mail: ishwarlal.jialal{at}ucdmc.ucdavis.edu.
ABSTRACT
Background: Oxidative stress and inflammation are crucial in atherogenesis. -Tocopherol is both an antioxidant and an antiinflammatory agent.
Objective: We evaluated the effect of RRR--tocopherol supplementation on carotid atherosclerosis in patients with stable coronary artery disease (CAD) on drug therapy.
Design: Randomized, controlled, double-blind trial compared RRR--tocopherol (1200 IU/d for 2 y) with placebo in 90 patients with CAD. Intimal medial thickness (IMT) of both carotid arteries was measured by high-resolution B-mode ultrasonography at 0, 1, 1.5, and 2 y. At 6-mo intervals, plasma -tocopherol concentrations, C-reactive protein (CRP), LDL oxidation, monocyte function (superoxide anion release, cytokine release, and adhesion to endothelium), and urinary F2-isoprostanes were measured.
Results: -Tocopherol concentrations were significantly higher in the -tocopherol group but not in the placebo group. High-sensitivity CRP concentrations were significantly lowered with -tocopherol supplementation than with placebo (32%; P < 0.001). -Tocopherol supplementation significantly reduced urinary F2-isoprostanes (P < 0.001) and monocyte superoxide anion and tumor necrosis factor release compared with baseline and placebo (P < 0.001). No significant difference was observed in the mean change in total carotid IMT in the placebo and -tocopherol groups. In addition, no significant difference in cardiovascular events was observed (P = 0.21).
Conclusions: High-dose RRR--tocopherol supplementation in patients with CAD was safe and significantly reduced plasma biomarkers of oxidative stress and inflammation but had no significant effect on carotid IMT during 2 y.
Key Words: Vitamin E • atherosclerosis • coronary artery disease • inflammation • oxidative stress • intimal media thickness • carotid
INTRODUCTION
Atherosclerosis is the leading cause of mortality in the Western world. Oxidative stress and inflammation are important in the pathogenesis of atherogenesis. Epidemiologic studies suggest an association between increased antioxidant intake, especially vitamin E, and reduced rates of morbidity and mortality from coronary artery disease (CAD) (1–5). Vitamin E is the most potent, lipid-soluble antioxidant in plasma. In addition, -tocopherol also has potential antiatherogenic effects on critical cells in atherogenesis such as monocyte-macrophages, smooth muscle cells, platelets, and endothelial cells (1–5). Vitamin E, especially RRR--tocopherol, exhibits antioxidant as well as antiinflammatory activity and inhibits several biological events involved in atherogenesis (1–6). Although the studies performed with cell culture and animal models suggest that -tocopherol has promising antiatherosclerotic effects, the results of its supplementation in humans in randomized prospective clinical trials were disappointing (7, 8). This could possibly be because of inadequate selection of subjects (by sex, vitamin E status, oxidative stress, etc) or the chemical form of tocopherol used (all-rac or RRR form) (5, 9).
Carotid atherosclerosis predicts CAD (10–13). Furthermore, studies suggest that both increased LDL oxidative susceptibility and low concentrations of -tocopherol correlate with carotid atherosclerosis (14, 15). Atherosclerosis in the carotid arteries can be reliably determined by measuring intimal medial thickness (IMT) with B-mode ultrasonography scans that have the clear advantage of being a noninvasive procedure and thus could be repeated frequently. Because there was a paucity of data examining the effect of high-dose RRR--tocopherol supplementation on carotid atherosclerosis in patients with CAD and on biomarkers of oxidative stress and inflammation, we tested the effect of high-dose RRR--tocopherol supplementation (1200 IU/d for 2 y) on carotid atherosclerosis, biomarkers of oxidative stress, and inflammation in patients with stable CAD in a placebo-controlled, randomized, double-blind trial.
SUBJECTS AND METHODS
Study design
This was a prospective, randomized, placebo-controlled, double-blinded trial initiated in 1999 to study the effects of 1200 IU RRR--tocopherol/d compared with placebo on the progression of carotid atherosclerosis in patients with CAD. The patients were following a National Cholesterol Education Panel Step II diet and taking cholesterol-lowering medications in the form of ß-hydroxy-ß-methylglutaryl coenzyme A reductase inhibitors (ie, pravastatin, simvastatin, lovastatin, atorvastatin).
Subjects
Patients with CAD (n = 90) were identified from the lipid and cardiology clinics and cardiac catheterization laboratories at the University of Texas Southwestern Medical Center (Dallas, TX) and the University of California Davis Medical Center (Sacramento, CA). These subjects were screened without restriction to sex, race, or socioeconomic status. Eligibility of the patients to participate in this study was based on the following criteria: age of 40–70 y; receiving hypolipidemic drug therapy for 1 y, and evidence of a prior myocardial infarction (>1 y after the event) or stable exertional angina. Exclusion criteria for the study included LDL cholesterol > 130 mg/dL, serum triacylglycerols > 350 mg/dL, liver enzymes > 2 times upper limit of the reference range, homocysteine > 15 µmol/L despite folate supplementation, untreated hypo- or hyperthyroidism, recent myocardial infarction (<6 mo), history of stroke or transient ischemic attacks, uncontrolled hypertension (blood pressure > 150/95 mm Hg on 2 visits) or congestive heart failure, malabsorption, treatment with corticosteroids or androgens, alcohol consumption [1 oz/d (30 mL/d to 24 g alcohol/d)], previous use of high-dose antioxidant supplements in the past 1 y, or likelihood of moving from the area in 3 y. Patients treated with thiazide diuretics were included if they were on a stable dose. Women taking oral contraceptive agents or hormone replacement therapy were included if they had been taking these medications for >1 y and were on a stable dose. The study was performed according to the responsible committee on human experimentation (University of Texas Southwestern Medical Center and the University of California, Davis, institutional review boards).
Methods
After obtaining informed consent, eligible subjects (n = 90) had 2 fasting blood samples drawn during the lead-in phase, with at least a 4-wk interval between each measurement. In addition, complete blood cell count, plasma lipid and lipoprotein profile, liver enzymes, creatinine, and homocysteine were measured with the use of standard laboratory techniques. Subjects were randomly assigned to receive either RRR--tocopherol (1200 IU/d) or a placebo (soybean oil; provided by Cognis Corporation, Cincinnati, OH) for the 2-y period. The IMT of both carotid arteries was measured by high-resolution B-mode ultrasonography scans at 0, 1, 1.5, and 2 y.
Bilateral B-mode ultrasonography scanning was performed as described previously (16, 17). Briefly, the subject is placed in a supine position with the head slightly rotated and the neck extended and relaxed. The sonographer is seated at the end of the examination table near the subject's head. The carotid arteries are examined bilaterally in the distal common carotid artery (1 cm proximal to the carotid bulb), the carotid bulb, and the proximal internal carotid artery (1 cm distal to the flow divider). The objective was to obtain images of the segment of vessel that contains the maximum wall thickness or abnormality, either near or far wall, in any one view. Three images were obtained at the site of interest from the 3 different scanning angles (the anterior-oblique, the lateral, and the posterior-oblique) to provide as much circumferential information at the site of maximum wall thickness as possible.
Near wall and far wall interfaces were measured according to the techniques described by Pignoli et al (16) in 6 sites along the distal common bulb and internal carotid arteries. All examinations were recorded on super VHS by 2 sonographers. All ultrasonography scans were read by Dr R Tang at Wake Forest University in a blinded fashion.
IMT progression rates were determined for each of the sites with the use of a least-squares regression line relating IMT measurements for each time point during the 2-y supplementation period. Progression of atherosclerosis was measured by 1) the mean change in IMT between the baseline and after the 2-y supplementation period and 2) rate of IMT progression.
Fasting blood and 24-h urine specimens were obtained at 0, 6 mo, 1 y, 1.5 y, and 2 y of supplementation. Pill counts were done at each period, and compliance by pill count was found to be high (91%). Monocytes were isolated from heparinized blood after Ficoll density centrifugation as described previously (18). Biomarkers of oxidative stress that were assessed included monocyte superoxide anion release by the ferricytochrome C method as described previously (18), urinary F2-isoprostanes (19), and copper-induced LDL oxidation (18). Biomarkers of inflammation that were assessed included high-sensitivity C-reactive protein (hsCRP) concentrations (20) and monocyte cytokines and monocyte adhesion to endothelium as described previously (18).
Statistics
All statistical analyses were performed by the University of Texas Southwestern General Clinical Research Center biostatistician with the use of SAS statistical software version 9.1 (SAS Institute, Cary NC). The primary endpoint was the progression rate in IMT (negative for regression or improvement, positive for progression). The study was powered (0.80) to detect a difference in IMT between groups of 0.02 mm with an estimated SD of 0.03 mm (type II error of < 0.20). Baseline characteristics were summarized with descriptive statistics, and 2-sample t tests or Fisher's exact tests were used to assess differences between groups.
Random coefficient models were used to compute the rate of progression for IMT and also to assess the effect of -tocopherol supplementation. A 2-factor repeated-measures analysis of variance with one repeated factor (responses during the 2 y of treatment) and one grouping factor (placebo compared with -tocopherol) was performed to assess changes in continuous variables (IMT measures, antioxidant concentrations, markers of oxidative stress, and inflammation). Multiple comparisons were made with contrasts from the analysis of variance models with = 0.01 for post hoc to adjust for multiple testing, and Bonferroni correction was used as necessary. Skewed variables were rank-transformed before analysis. Spearman's correlation was performed to assess the relation between changes in plasma vitamin E concentrations and other variables of interest.
RESULTS
Baseline subject characteristics are reported in Table 1. In addition, as reported recently, -tocopherol concentrations were significantly higher in the -tocopherol group but not in the placebo group, and no changes were observed in the lipid profile in patients receiving RRR--tocopherol compared with placebo (Table 2) (21). However, plasma -tocopherol concentrations were significantly higher in the RRR--tocopherol group than in the placebo group (P < 0.001, time-by-treatment interaction; Table 2) (21). In addition, during the 2-y period, RRR--tocopherol supplementation did not result in any changes in blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, thyroid-stimulating hormone, complete blood cell count, including white blood cell and platelet counts (data not shown).
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TABLE 1. Baseline characteristics of patients in the study1
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TABLE 2. Effect of RRR--tocopherol supplementation on lipid profile and plasma -tocopherol concentrations1
During the 24-mo study period, hsCRP concentrations were significantly lower with -tocopherol supplementation than with placebo (32%; P < 0.001, time-by-treatment interaction; Figure 1). For monocyte cytokines, a time-by-treatment interaction effect for significant reduction was observed with tumor necrosis factor- (TNF-) (P < 0.001, time-by-treatment interaction; Table 3). Monocyte-endothelial cell adhesion was not significantly different with high-dose RRR--tocopherol supplementation than with placebo (Table 3).
FIGURE 1.. Effect of RRR--tocopherol supplementation on median high-sensitivity C-reactive protein (hsCRP). hsCRP concentrations were tested at baseline and 6, 12, 18, and 24 mo after RRR--tocopherol (1200 IU/d) or placebo as described in "Subjects and Methods." Median and interquartile range are shown. P < 0.001, time-by-treatment interaction (repeated-measures ANOVA); #significantly different from baseline, P < 0.0001.
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TABLE 3. Effect of -tocopherol supplementation on monocyte cytokines and monocyte-endothelial cell adhesion after lipopolysaccharide activation1
For biomarkers of oxidative stress, -tocopherol supplementation significantly reduced LDL oxidizability as measured by the increase in lag time of LDL oxidation (P < 0.001, time-by-treatment interaction; Figure 2), urinary F2-isoprostanes (P < 0.001, time-by-treatment interaction; Figure 2), and monocyte superoxide anion concentrations than did placebo (P < 0.001, time-by-treatment interaction; Figure 2).
FIGURE 2.. Effect of RRR--tocopherol supplementation on LDL oxidizability, urinary F2-isoprostanes, and monocyte superoxide anion release. LDL oxidizability, assessed as lag time of LDL oxidation; F2-isoprostanes, measured in 24-h urine specimens, and monocyte superoxide anion release after lipopolysaccharide activation (1 µg/mL) were assayed at baseline and 6, 12, 18, and 24 mo after RRR--tocopherol (1200 IU/d) or placebo as described in "Subjects and Methods." Data are shown as means and SDs. LDL oxidizability, F2-isoprostanes, and monocyte superoxide anion were significantly reduced with -tocopherol supplementation compared with placebo (P < 0.001, time-by-treatment interaction; repeated-measures ANOVA); #significantly different from baseline, P < 0.0001.
No significant difference was observed in the mean change in total carotid IMT between the placebo and RRR--tocopherol groups (P = 0.17, time-by-treatment interaction; Table 4). Cardiovascular events (including myocardial infarction, stroke, bypass surgery, percutaneous coronary angioplasty, transient ischemic attack, angina, and death) were not significantly different in the RRR--tocopherol group than in the placebo group (16% compared with 28%; P = 0.21). Significant correlations were found between changes in plasma vitamin E concentrations (change of 2 y – baseline) compared with plasma CRP (r = –0.46, P < 0.0001), F2-isoprostanes (r = –0.57, P < 0.0001), lag time of LDL oxidation (r = 0.70, P < 0.0001), monocyte superoxide anion release (r = –0.57, P < 0.0001), and TNF- (r = –0.57, P < 0.0001).
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TABLE 4. Effect of -tocopherol supplementation on carotid atherosclerosis in patients with coronary artery disease1
DISCUSSION
Strategies that lower LDL cholesterol, lower blood pressure, and provide antiplatelet therapy reduce cardiovascular events (22–24). Although statins have been one of the most effective therapies in the prevention of cardiovascular disease, the collaborative meta-analyses of all trials show only a 21% reduction in cardiovascular events (25). Thus, there is a clear need for additional therapies to prevent cardiovascular disease. Most studies with vitamin E (-tocopherol) have yielded a null result (1–9). Many of those studies have been criticized because, although a large number of subjects were studied, the populations may not have had an increased burden of oxidative stress; relevant biomarkers of oxidative stress and plasma concentrations of antioxidants such as -tocopherol were not reported (1–9). Furthermore, studies did not consistently use RRR--tocopherol, the form shown to be an antioxidant and an antiinflammatory; when used, the doses of RRR--tocopherol have not been sufficiently high (9). Thus, the present study was undertaken in a high-risk population with increased oxidative stress, ie, patients with stable CAD using a dose of -tocopherol (1200 IU/d) that was shown previously to prevent lipid peroxidation and to induce antiinflammatory effects (9). In addition, the present study examined plasma concentrations of -tocopherol, biomarkers of oxidative stress and inflammation, and carotid atherosclerosis. In this comprehensive study, we clearly show that RRR--tocopherol supplementation resulted in significantly higher plasma concentrations of -tocopherol and significantly lowered biomarkers of oxidative stress (F2-isoprostanes and LDL oxidative susceptibility) and inflammation (a significant reduction in hsCRP, monocyte superoxide, and TNF concentrations). However, compared with placebo, -tocopherol supplementation did not result in any significant change in common carotid artery IMT. In addition, as Meydani et al (26) have reported previously in a 4-mo study, we also failed to observe any adverse effects on kidney, renal, thyroid function tests; lipid profile; and complete blood cell count with -tocopherol supplementation.
Other studies that have evaluated the effect of
Our study did not have sufficient statistical power to assess the effect of RRR--tocopherol on cardiovascular events, which was not a predefined endpoint. However, because of the high-risk population, cardiovascular events were documented. Previously, it was shown that concomitant reduction of CRP and LDL cholesterol in the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE-IT) study resulted in the greatest benefit for cardiovascular events (30). Furthermore, a reduction in CRP to <2 mg/L was associated with benefit at all concentrations of LDL cholesterol achieved. In the present study, 25% of patients in the -tocopherol group achieved a reduction in CRP to <2 mg/L. Because vitamin E had no beneficial or deleterious effect on the lipid profile or other safety measures in this study as reported previously (21) but did reduce CRP concentrations consistently and cumulatively with time, it is possible that the longer administration of the combination of vitamin E with statins in a study with larger sample size could possibly yield a further reduction in cardiovascular events in patients with acute coronary syndromes. Murphy et al (31) have also shown that in smokers with acute coronary syndromes vitamin E lowers CRP concentrations. That hypothesis can only be tested in a future trial that evaluates cardiovascular events in patients with acute coronary syndromes. In the Women's Health study (32), with the longest duration (average: 10.1 y), RRR--tocopherol supplementation (600 IU/d on alternate days) in participants 65 y of age resulted in a 20% reduction in cardiovascular events that was significant (P < 0.01); it might not be unreasonable to consider conducting a trial with a combination of statin and -tocopherol in such a group. Furthermore, it appears that of the different biomarkers of inflammation CRP appears to be the most robust to assess response over a longer duration. Notably, a progressive decrease in CRP concentrations with time was observed in the -tocopherol group (reductions from baseline at 6, 12, 18, and 24 mo were 4%, 16%, 29%, and 33%, respectively). Although we have previously shown in short-term studies that high-dose RRR--tocopherol significantly lowers proinflammatory cytokine release compared with placebo, there was a reduction with only TNF with -tocopherol supplementation compared with placebo during the 2-y period. Thus, these markers did not hold with the longer duration of supplementation as performed in this study.
Certain lessons can be gleaned from the present study. As shown in the VEAPS study and other studies, lowering LDL-oxidative susceptibility does not equate to a significant reduction in atherosclerosis or cardiovascular events, and it might not be a relevant biomarker for increased oxidative stress. Although reduction in isoprostanes in our study did not translate to reduced IMT progression with -tocopherol, in the ASAP study, in which they used a combination of vitamins E and C, they showed a reduction in F2-isoprostanes in men but not women, supporting that F2-isoprostanes are a valid biomarker of oxidative stress.
Although statins are shown, in addition to LDL cholesterol lowering, to have pleiotropic antiinflammatory effects, resulting in decreased cardiovascular events (33), we propose here that antioxidants such as vitamin E may not produce the desired reduction in cardiovascular events and atherosclerotic burden, despite a reduction in biomarkers of oxidative stress and inflammation, possibly because of a lack of dual effect on the lipid profile and CRP concentrations. Because -tocopherol was suggested to have superior antioxidant and antiinflammatory effects, consideration should be given to combined - and -tocopherol supplementation because -tocopherol supplementation decreases -tocopherol concentrations (34, 35).
In conclusion, although this study confirms that RRR--tocopherol has no benefit on carotid atherosclerosis, it establishes that hsCRP and urinary F2-isoprostanes are valid and robust biomarkers during a 2-y period and that RRR--tocopherol supplementation was safe. It needs to be noted, however, that in other populations at increased risk, such as patients with nonalcoholic steatohepatitis, vitamin E may still be beneficial (36), and its use is being tested in randomized clinical trials such as the PiVENS (Pioglitazone versus Vitamin E versus Placebo for the Treatment of Nondiabetic Patients with Nonalcoholic Steatohepatitis) and TONIC [Treatment of Nonalcoholic Fatty Liver Disease (NAFLD) in Children] trials of patients with nonalcoholic steatohepatitis.
ACKNOWLEDGMENTS
We thank the cardiologists at the University of Texas Southwestern Medical Center and the University of California, Davis, Medical Center for help with recruitment of patients for study.
The author's responsibilities were as follows—AH and TS: helped with study coordination; BA-H: analyzed statistical data; JAdeL: follow up of patients at University of Texas Southwestern Medical Center; RT: read IMT tapes at Wake Forest University; SD (study coordinator): helped with study analyses and manuscript preparation; IJ: overall follow-up of patients, direction of study, and manuscript preparation. None of the authors had a personal or financial conflict of interest.
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