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J Am Coll Cardiol, 2004; 44:1996-2002, doi:10.1016/j.jacc.2004.08.029 © 2004 by the American College of Cardiology Foundation |
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* Elucida Research, Beverly, Massachusetts
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Pfizer Inc., New York, New York
Pfizer Central Research, Groton, Connecticut
Manuscript received July 9, 2004; revised manuscript received August 3, 2004, accepted August 10, 2004.
* Reprint requests and correspondence: Dr. R. Preston Mason, 100 Cummings Center, Suite 135L, Beverly, Massachusetts 01915 (Email: rpmason{at}elucidaresearch.com).
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Abstract |
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BACKGROUND: Oxidative stress contributes to mechanisms of atherosclerosis and plaque instability. Biomarkers of oxidation, such as malondialdehyde (MDA), may represent independent indicators of risk for patients with stable coronary artery disease (CAD).
METHODS: Serum MDA levels were measured as thiobarbituric acid reactive substances (TBARS) in 634 patients with documented CAD using reverse-phase high-performance liquid chromatography and spectrophotometric approaches.
RESULTS: During the three-year study, there were 51 major vascular events such as fatal/nonfatal myocardial infarction, 149 hospitalizations for nonfatal vascular events, and 139 patients underwent a major vascular procedure. At baseline, patients with TBARS levels in the highest quartile had a relative risk (RR) of 3.30 (95% confidence interval [CI] 1.47 to 7.42; p = 0.038) for major vascular events, RR of 4.10 (95% CI 2.55 to 6.60; p < 0.0001) for nonfatal vascular events, and RR of 3.84 (95% CI 2.56 to 5.76; p < 0.0001) for major vascular procedures. The effect of TBARS on events and procedures was also seen in a multivariate model adjusted for inflammatory markers (C-reactive protein, soluble intercellular adhesion molecule-1, interleukin-6), and other risk factors (age, low-density lipoprotein, high-density lipoprotein, total cholesterol, triglycerides, body mass index, and blood pressure). This analysis showed an independent effect of TBARS on major vascular events (p = 0.0149), nonfatal vascular events (p < 0.0001), major vascular procedures (p < 0.001), and all vascular events and procedures (p < 0.0001).
CONCLUSIONS: Serum levels of TBARS were strongly predictive of cardiovascular events in patients with stable CAD, independently of traditional risk factors and inflammatory markers.
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The hypothesis of the current study is that elevated levels of TBARS are associated with increased cardiovascular risk in patients with stable coronary artery disease (CAD). This hypothesis was tested using serum samples obtained from The Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT), a prospective, double-masked clinical trial. Patients in this study had documented coronary disease and were treated with either amlodipine or placebo. Treatment with amlodipine was associated with significantly fewer cases of unstable angina and coronary revascularization without a reduction in luminal loss (11). This benefit with amlodipine may be attributed to various mechanisms, as previously reviewed (12). For the biomarker analysis, serum samples were collected from 634 patients at baseline and at the end of each 12-month period during the 3-year study. The level of the MDA-thiobarbituric-acid complex was precisely measured after separation by reverse-phase high-performance liquid chromatography coupled with spectrophotometric and fluorescence detection (13,14). The association of TBARS with clinical events and procedures was evaluated in univariate and multivariate models adjusting for inflammatory markers (C-reactive protein [CRP], soluble intercellular adhesion molecule-1 [sICAM-1], interleukin-6 [IL-6]), and other risk factors (age, gender, lipids, body mass index, and blood pressure).
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Methods |
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30% diameter stenosis (non-intervened and non-infarcted) and the presence of 1 lesion with a 5% to 20% stenosis that was not in a vessel with a 60% lesion. Other eligibility criteria included diastolic blood pressure of <95 mm Hg, total cholesterol of <325 mg/dl, and fasting blood glucose of <200 mg/dl. Randomization was stratified according to clinical center and history of percutaneous transluminal coronary angioplasty (PTCA). Monitoring for clinical events and adverse experiences. The prespecified clinical events were all-cause mortality and the occurrence of major fatal/nonfatal vascular events or procedures. Death, myocardial infarction, stroke, hospitalized heart failure, and hospitalized episodes of unstable angina were classified by an external events classification committee blinded to treatment assignment. Confirmation of unstable angina required hospitalization for typical chest pain and either evidence of myocardial ischemia (electrocardiogram or stress test evidence, or new angiographic findings of disease) or an indication that this pain was similar to that of previously documented evidence of ischemia.
Angiographic methods and outcomes.
The progression of early atherosclerotic segments was determined on the basis of a change in mean minimal diameter with quantitative coronary angiography (QCA) (15,16). Atherosclerotic segments were defined as coronary segments with a diameter stenosis of 
30% at baseline. Up to 12 coronary segments were used in the analysis of disease progression (17). Vessels that underwent a procedure at or before baseline were excluded from the analyses. The baseline and follow-up films were centrally read pairwise by a certified reader who was blinded to treatment assignment and the temporal sequencing of films.
Measurement of serum TBARS levels. Serum samples were obtained from 634 fasting participants at the beginning of the study (baseline) and at the end of each of the three years. Samples were stored at –70°C without the addition of exogenous antioxidants before TBARS analysis. After thawing the samples, measurements of MDA in terms of TBARS were performed in duplicate in each of 2,975 samples by the method of Carbonneau et al. (18) with slight modifications. Briefly, 50 µl of 10 M sodium hydroxide (NaOH) was added to 0.5 ml of serum and incubated at 60°C for 30 min. The sample was then acidified to pH 1.0 with 500 µl of 586 g/l perchloric acid. After centrifugation, 300 µl of supernatant was added to 50 µl of thiobarbituric acid or TBA (10 g/l in 50 mM phosphate buffer, pH 7.0) and heated at 100°C for 30 min. The sample was cooled and 100 µl removed for high-performance liquid chromatography analysis. The MDA that reacts with TBA in this reaction is generated from lipid hydroperoxides and potentially other biologic sources (e.g., glucose).
The MDA-thiobarbituric-acid complex was separated from other possible reactants with TBA using reverse-phase high-performance liquid chromatography on a Varian Prostar system (Varian Inc., Walnut Creek, California)coupled with a spectrophotometric detector at 532 nm and a fluorescence detector (excitation = 515 nm, emission = 553 nm) on a 150 mm x 4.6 mm Adsorbosphere (Alltech Associates Inc., Deerfield, Illinois) C18 column with 5 µm particle size. The purpose for using reverse-phase high-performance liquid chromatography followed by quantitation with both spectrophotometry and fluorescence was to eliminate other aldehydes that react with TBA and have absorbance characteristics at 532 nm (13,14,19). The flow rate was 1 ml/min, and the mobile phase was 80% phosphate buffer (10 mM, pH 5.8) with 20% methanol. A standard curve was run at the start, middle, and end of each sample set using 1,1,3,3-tetraethoxypropane as a standard. Peak areas were determined using the Star Chromatographyworkstation (Varian Inc.).
Measurement of serum CRP, IL-6, and sICAM-1. C-reactive protein levels were measured using the N Latex CRP mono assay (Dade Behring Inc., Deerfield, Illinois) with a detection limit of 0.21 mg/l. Samples with values below the limit of detection were recorded as <0.21 mg/l; the value 0.20 mg/l was incorporated for statistical analyses. Intra- and interassay precision for the low quality control (QC)(0.46 mg/l) had coefficient of variations (CVs)of 9.9% and 14.8%, respectively. Serum soluble intercellular adhesion molecule-1 levels were measured with the Parameter Human sICAM-1 Immunoassay Kit (R&D Systems, Minneapolis, Minnesota), with a range of 0 to 588 ng/ml. Intra- and interassay precision for the middle QC (282.7 ng/ml) had CVs of 9.0% and 9.5%, respectively. Interleukin-6 levels were measured using the Quantikine HS IL-6 R&D Systems kit, which had an assay range of 0.156 to 10 pg/ml. Intra- and interassay precision for the low QC (0.338 pg/ml) had CVs of 9.9% and 14.4%, respectively. All measurements were made by Esoterix Coagulation (Aurora, Colorado).
Statistical analysis. Simple descriptive statistics were used to describe the population. For clinical outcomes, proportional hazards regression models were used to obtain hazards ratios and associated 95% confidence intervals (CI). The first proportional hazards model used baseline TBARS and treatment as covariates for each clinical outcome (major vascular events, hospitalizations for angina, coronary artery bypass grafting [CABG], PTCA, and major vascular procedures). Finally, to further investigate the impact of TBARS on clinical outcomes, a proportional hazards model was performed using quartiles of baseline TBARS with the reference group being those in the lowest quartile.
Pearson's correlation coefficients were used to assess the correlation between TBARS and coronary angiography outcomes measurements (e.g., all segments, segments stenosed 30%, segments stenosed <30% and 50%, and segments stenosed >50%) as well as TBARS and changes in patient characteristics (e.g., change in systolic blood pressure, change in diastolic blood pressure, change in high-density lipoprotein [HDL], change in LDL, and change in triglycerides). All analyses were undertaken using alpha = 0.05 and were performed using SAS Version 8.2 (SAS Institute Inc., Cary, North Carolina).
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TBARS levels and clinical events. Table 2 shows the relationship between measured baseline levels of TBARS and cardiovascular events in PREVENT. At baseline, the overall mean absolute level of TBARS was 1.49 ± 0.57 µM. Baseline levels of TBARS were associated with risk for fatal/nonfatal myocardial infarction with a relative risk (RR) of 2.94 (95% CI 1.75 to 4.94), an RR of 2.58 (95% CI 1.98 to 3.37) for nonfatal cardiovascular events, and an RR of 2.14 for nonfatal vascular events (95% CI 1.61 to 2.84). The absolute levels of TBARS in patients with and without specific vascular events and procedures events were reviewed in Table 3. The significant univariate effect of TBARS seen on major vascular events, nonfatal vascular events, vascular procedure, and all vascular events and procedures is also seen in a multivariate model adjusting for inflammatory markers and known cardiovascular risk factors. Specifically, a multivariate Cox proportional hazards regression model was undertaken where the following variables were included in the analysis: TBARS, inflammatory markers (sICAM-1, IL-6, CRP), age, gender, total cholesterol, HDL, LDL, triglycerides, systolic blood pressure, diastolic blood pressure, and body mass index. After adjusting for all of these variables, TBARS levels showed an independent effect on major vascular events (p = 0.0149), nonfatal vascular events (p < 0.0001), vascular procedure (p < 0.001), and all vascular events and procedures (p < 0.0001).
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In Figure 1, it is demonstrated that participants with the highest baseline TBARS levels were at increased risk for experiencing a major vascular event (RR of 3.30; 95% CI 1.47 to 7.42), nonfatal cardiovascular event (RR of 4.10; 95% CI 2.55 to 6.60), major vascular procedure (RR of 3.29; 95% CI 2.09 to 5.15), and all vascular events and procedures (RR of 3.84; 95% CI 2.56 to 5.78). Additionally, patients in the highest quartile had an increased risk of fatal/nonfatal MI (RR of 5.07; 95% CI 1.70 to 15.06; p = 0.0035), developing angina (RR of 4.03; 95% CI 2.50 to 6.49; p < 0.0001), undergoing a CABG procedure (RR of 3.03; 95% CI 1.33 to 6.88; p < 0.0080) or PTCA (RR of 3.12; 95% CI 1.74 to 5.58; p < 0.0001), and experiencing a fatal/nonfatal myocardial infarction (RR of 5.07; 95% CI 1.70 to 15.06; p = 0.0035).
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The mean absolute levels of TBARS were stable over the three-year period due to uniformity in the handling and storage of the samples. The mean absolute levels were 1.5 ± 0.6 µM, 1.4 ± 0.3 µM, 1.3 ± 0.4 µM, and 1.3 ± 0.4 µM at baseline, month 12, month 24, and month 36, respectively. By contrast, the absolute mean levels of TBARS in the highest quartiles were in the range of 3.3 to 4.1 µM, depending on the cardiovascular event category.
TBARS levels and angiographic measurements..
Changes in TBARS over the three-year study correlated poorly with angiographic outcome measurements. In all segments evaluated, there was a significant association (n = 515; p = 0.021) between angiographic determinations of lesion development and serum TBARS levels, but the correlation coefficient was 0.100. In particular, changes in TBARS levels correlated with progression of moderate coronary stenoses (>30% and 50%) in a significant fashion (n = 474; p = 0.024) with a correlation coefficient of 0.105, but not in minimal segments of 30% (n = 507; p = 0.085) or larger segments of 50% (n = 237; p = 0.742). The p values were calculated from the Spearman rank correlation coefficient.
TBARS levels and patient characteristics. Thiobarbituric acid reactive substances chromatography levels were strongly associated with a higher risk of cardiovascular morbidity, independently of other known risk factors, such as blood pressure (systolic and diastolic) or lipids (HDL, LDL, and triglycerides). Levels of TBARS also did not correlate with baseline demographics such as gender, history of smoking, previous angina, or a family history of myocardial infarction or sudden death. However, a patient's history of a myocardial infarction correlated significantly with baseline TBARS levels (n = 659; p < 0.035). The level of TBARS was 1.54 ± 0.61 µM (n = 309) for patients who had a history of a myocardial infarction, whereas the level was 1.45 ± 0.52 µM (n = 350) for patients without such a previous event.
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Discussion |
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50%). Although amlodipine treatment was associated with significantly fewer cardiovascular events, it did not influence TBARS levels. To our knowledge, this is the first longitudinal study demonstrating an association between elevated TBARS in serum and cardiovascular risk in patients with stable CAD. Previous studies in relatively small populations had demonstrated correlations between levels of TBARS and key cardiovascular risk factors, including cigarette smoking, hypertension, hyperlipidemia, and diabetes (5,6). Markers of lipid peroxidation, including LDL oxidation, have also been associated with the development of coronary disease (20–24). In one study of 206 subjects, TBARS levels correlated with severity of disease as determined by angiography, independently of other known risk factors (total cholesterol, LDL, triglycerides) (21). In another small study of 29 patients with coronary disease, use of lipid-lowering agents (HMG-CoA reductase inhibitor, bile sequestrant) was associated with significantly lower levels of serum TBARS in a manner that correlated strongly with endothelial function (25). Collectively, these studies support an emerging role for markers of oxidative stress in the prediction of risk for cardiovascular disease.
In this study, the serum levels of TBARS measured at baseline in these patients correlated directly with levels of the proinflammatory cytokine, IL-6. Production of IL-6 from vascular cells is triggered by the proatherogenic factor, angiotensin II (26), which also stimulates reactive oxygen species involved in LDL oxidation. Cytokine production from smooth muscle cells is also stimulated directly by oxidized LDL (27). Given the interrelationships between cytokine expression, angiotensin II, and reactive oxygen species, the correlation between levels of this cytokine and TBARS may result from common early events in atherogenesis. Elevations in IL-6, in turn, lead to increased release of the acute phase reactant CRP from the liver (28), an inflammatory marker that predicts cardiovascular risk (29). The lack of an association between levels of TBARS and CRP is not well understood but may indicate only an indirect relationship between the expression of certain inflammatory markers and markers of advanced oxidative damage in these patients with stable CAD.
Oxidative modification of lipids associated with LDL and cellular constituents contribute to endothelial dysfunction and inflammatory pathways associated with atherosclerosis (1–3,30,31). The mechanism of in vivo LDL oxidation is not fully understood, but results of mass spectroscopy analyses indicate elevated levels of protein oxidation products in human atherosclerotic lesions that may be attributed to myeloperoxidase activity (32). Besides measurements of TBARS, other approaches have been used to assess oxidative stress levels, such as monoclonal antibodies against oxidized LDL, protein oxidation markers, and measurement of isoprostanes (33–35). Isoprostanes are prostaglandin-like compounds that may be formed in vivo from the free-radical-initiated peroxidation of arachidonic acid, independently of the cyclooxygenase pathway. Studies have demonstrated that these compounds are found in the plaque and associated with increased risk for cardiovascular disease (36,37). In vivo oxidative stress levels have been quantitated by several different methods (33,38,39).
Study limitations. A limitation of this study was that the MDA measured in the TBARS assay may be generated, in some part, from sources other than lipid hydroperoxides associated with lipoproteins, such as glucose, bilirubin, and amino acids. Additionally, measurements were carried out with serum samples from patients, the preparation of which could theoretically lead to some MDA development. Finally, preservatives (e.g., antioxidants) were not added to the serum samples as part of the storage procedure at –70°C, and this could contribute to MDA formation over time. It should be noted, however, that the absolute mean levels of MDA were stable over the course of the three-year study.
Implications for antioxidant treatment. The implication of these data for the role of antioxidants in the treatment of CAD is an interesting question. Although epidemiologic and laboratory animal studies indicate that low levels of antioxidants are associated with increased risk for cardiovascular disease, the results of large prospective antioxidant clinical trials have failed to show a benefit, as recently reviewed (40,41). Possible explanations for this paradox may be due to trial design, baseline antioxidant status of participants, dosage and source of the antioxidants, and time of intervention relative to disease progression. An additional explanation is that vitamin E does not neutralize relevant oxidants, such as those produced by myeloperoxidase (42), and/or there is reduced penetration of this natural antioxidant into the atherosclerotic plaque. This latter concept was evaluated in patients with advanced atherosclerosis after vitamin E supplementation (450 IU/day). This analysis showed that, despite a reduction in plasma markers of oxidation with vitamin E, there was no change in vitamin E content or levels of oxysterols in the plaque itself (43). This may be due to the effects of hyperlipidemia on the cholesterol content on vascular cell plasma membranes (44). This increase in membrane cholesterol content interferes with the ability of lipophilic molecules to partition into the lipid environment, as we have previously demonstrated with cardiovascular agents (45). Thus, vessel wall changes in patients with advanced atherosclerosis may attenuate the ability of tocopherol to partition into plaque and scavenge free radicals. Synthetic antioxidants with superior scavenging activity and lipophilic properties may have different effects on oxidative stress markers in the plaque and beneficially influence the course of the disease.
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