Clinical Predictors of Oxidized Low-Density Lipoprotein in Patients With Coronary Artery Diseasefn1

Clinical Predictors of Oxidized LowDensity Lipoprotein in Patients With Coronary Artery Disease Lori Mosca, MD, MPH, PhD, Melvyn Rubenfire, MD, Tom Tarshis, Alan Tsai, PhD, and Thomas Pearson, MD, MPH, PhD

MPH,

Oxidized low-density lipoprotein (LDL) may play a key role in the initiation and progression of atherosclerosis. Risk factors for elevated levels of oxidized LDL are not well established and may be important in identifying individuals who may benefit from antioxidant supplementation or interventions to reduce oxidant stress. The purpose of this study was to determine if clinical parameters predict levels of oxidized LDL. We evaluated the relation between clinical parameters and oxidized LDL in 45 nonsmoking, nondiabetic patients (39 men and 6 women) with coronary artery disease. Oxidized LDL was assessed by measurement of conjugated dienes, lipid peroxides, and thiobarbituric reactive substances (TBARS) at 0 hours to evaluate baseline oxidant stress

and postincubation with an oxidizing agent to assess the capacity of LDL for peroxidation. Results were lipid standardized and were not materially altered by multivariate adjustment. Significant predictors of increased oxidized LDL included female sex, family history of premature cardiovascular disease, increased percent body fat, increased body mass index, increased heart rate at rest, history of smoking, exercise <4 times per week, and no regular wine consumption. These data suggest that clinical parameters correlate with levels of oxidized LDL and may be useful in identifying patients at risk for increased oxidant stress. Q1997 by Excerpta Medica, Inc. (Am J Cardiol 1997;80:825– 830)

isk factors for elevated levels of oxidized lowR density lipoprotein (LDL) are not well established and it is unclear whether patients without increased

May 1995. Coronary artery disease was documented by angiogram (21 lesion .70% luminal diameter stenosis), prior angioplasty, coronary artery bypass surgery, myocardial infarction by history validated by electrocardiographic changes, or classic angina with a ischemic response on a treadmill test. To be included subjects also had to report a stable diet and exercise pattern for 6 months. Exclusion criteria included tobacco use within 6 months, abnormal hepatic or renal function, evidence of malabsorption, pancreatic or biliary disease, use of multivitamin supplements exceeding the recommended daily allowance, or any acute medical condition within 3 months of the study. All subjects gave informed consent. The study was approved by the Institutional Review Board of The University of Michigan Medical Center. Laboratory measurements: Following a 12-hour fast and a 15-minute resting period, blood was obtained by venipuncture with tourniquet deflated. Determination of LDL oxidation was performed on fresh serum cooled up to 6 hours. Lipid and lipoprotein measurements were carried out on serum stored in screw top tubes frozen at 215°C for up to 2 weeks. Measurements were performed on duplicate samples from each subject obtained 1 week apart and were averaged for statistical analysis. LDL lipid peroxidation was assessed by measurement of conjugated dienes, lipid peroxides, and thiobarbituric acid reactive substances (TBARS). Measurements were performed at 0 hour and postincubation with 3-mM copper chloride at 37°C for 2 hours to induce oxidation. Zero-hour levels reflect oxidant status. The 2-hour value represents the capacity for peroxidation. The susceptibility of LDL to oxidation was evaluated by measurement of lag phase. Detailed laboratory methodology is described in the following.

levels of oxidized LDL will have a favorable risk and cost-benefit ratio associated with antioxidant supplementation. Patients with angina have been shown to have increased levels of oxidized LDL compared with controls.1–3 Diabetes mellitus may worsen lipoprotein oxidation,4 and smokers have lower levels of plasma antioxidants than controls.5 Few data are available to evaluate what other risk factors may identify patients at risk for increased oxidized LDL. We undertook the present study to determine whether certain clinical parameters were associated with levels of oxidized LDL among patients with established coronary artery disease.

METHODS

Subjects: Forty-five nonsmoking, nondiabetic subjects with established coronary artery disease (39 men and 6 women), aged 39 to 80 years, were recruited from the Cardiac Catheterization Laboratory and the Preventive Cardiology Program at the University of Michigan Medical Center between January 1994 and From the Preventive Cardiology Program, Division of Cardiology, Department of Medicine and Human Nutrition Program, School of Public Health, University of Michigan, Ann Arbor, Michigan; and Department of Community and Preventive Medicine, University of Rochester, Rochester, New York. Dr. Mosca is the recipient of Scientist Development Grant 9636321W from The American Heart Association. The project was sponsored by a gift from Harold and Kay Peplau to the University of Michigan Preventive Cardiology Program. Manuscript received April 23, 1997; revised manuscript received and accepted June 17, 1997. Address for reprints: Lori Mosca, MD, MPH, PhD, Preventive Cardiology Program, The University of Michigan, 24 Frank Lloyd Wright Drive, P.O. Box 363, Ann Arbor, Michigan 48106-0363. ©1997 by Excerpta Medica, Inc. All rights reserved.

0002-9149/97/$17.00 PII S0002-9149(97)00530-4

825

For assessment of LDL oxidation parameters, LDL was isolated immediately from fresh serum according to the method of Weiland and Seidel.6 This method correlates well with results from ultracentrifugation in combination with polyanion precipitation (r 5 0.98) or quantitative lipoprotein electrophoresis (r 5 0.93). Quantitation of lipid peroxides, the major initial products of lipid peroxidation, were determined by the method of El-Saadani et al.7 This technique is based on the oxidative ability of lipid peroxides to convert iodide to iodine, which can be measured spectrophotometrically at 365 nm. Measurement of conjugated dienes was performed according to the method of Placer.8 The conjugation of double bonds of polyenoic fatty acids takes place at the first stage of lipid peroxidation reactions and can be measured spectrophotometrically at 234 nm. TBARS, which measures the end products of the peroxidative reaction (i.e., malondialdehyde), were determined according to the procedure of Naito et al.9 Malondialdehyde reacts with thiobarbituric acid during a heating process to produce substances that can be measured spectrophotometrically at 535 nm. For measurement of lag phase, the oxidation of polyunsaturated fatty acids in a buffer system was continuously monitored at 234 nm with a Beckman DU-6 Spectrophotometer (Fullerton, California) up to 6 hours according to the method of Esterbauer et al.10 Lag phase is expressed in minutes and is the time interval from the addition of Cu11 ions to the onset of the propagation phase of LDL oxidation, measured by dropping a tangent from the slope of the absorbance curve during the most rapid rise to the time axis for production of conjugated dienes. Lag phase is indicative of the resistance of LDL to oxidation; the greater the resistance of LDL to oxidation, the longer the lag phase. Cholesterol and triglyceride concentrations were determined enzymatically with commercially available reagents (Sigma, St. Louis, Missouri) by the methods of Allain et al11 for total, high-density lipoprotein (HDL), and LDL cholesterol and McGowan et al12 for total and LDL triglyceride. The isolation of LDL was according to the method of Weiland and Seidel.6 HDL was isolated by precipitation with phosphotungstic and/or magnesium chloride according to the method of Assmann et al.13 Measurement of covariates: Demographic variables, lifestyle habits, and medication usage were assessed by standardized questionnaire. Family history of premature heart disease was defined as myocardial infarction, coronary artery bypass surgery, angioplasty, sudden death, peripheral arterial disease, or cerebrovascular event in a first degree male relative ,55 years old or a female relative ,65 years old. Wine consumption was categorized as any versus no regular use of wine (at least one 4 ounce glass/week). Current vitamin use included daily intake of any vitamin or multivitamin supplement. Exercise level was quantified as the number of sessions of aerobic exercise per week resulting in a sweat. Anthropometric and physiologic variables were 826

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evaluated based on a standard examination that included measurement of height to the nearest 0.5 cm and weight to the nearest 0.1 kg without shoes in light clothing. Body fat was calculated from 3 sex-specific skinfold measurements using Jackson and Pollock generalized equations for predicting body density.14,15 Heart rate and blood pressure at rest were measured using standard American Heart Association protocols. Hypertension was defined as systolic blood pressure $140 mm Hg or diastolic blood pressure $90 mm Hg or current use of antihypertensive medications. Statistical analysis: Continuous variables are expressed as mean 6 SD. Normal probability plots of all continuous variables were examined and the ShapiroWilk Statistic was used to test for normal distribution. Log transformation was carried out where appropriate. LDL oxidation parameters were evaluated unadjusted and lipid adjusted by dividing by LDL cholesterol plus LDL triglyceride. The relations between continuous variables were evaluated using Pearson’s correlation coefficients or Spearman correlations when data were not normally distributed. Differences in means for dichotomous variables were examined by Student’s t tests or Wilcoxon rank sums for non-normally distributed variables. Significant independent predictors of LDL oxidation were incorporated into a multiple linear regression model to evaluate potential confounders. All tests were 2-sided and statistical significance was set at a ,0.05. All data were double entered, discrepancies corrected, and analysis performed using SAS for Windows version 6.08 (1992 SAS Institute, Inc, Cary, North Carolina).

RESULTS Characteristics of the 45 study participants are presented in Table I. The majority of subjects were men (87%) and white (96%). As a group, they had extensive coronary artery disease. Based on angiographic data in 31 subjects, 73% had 3-vessel disease by coronary angiography. As expected, the prevalence of cardiovascular risk factors in this group of cardiac patients was high. Use of b blockers and lipid-lowering drugs, 27% and 53%, respectively, was comparable to most coronary artery disease populations. LDL cholesterol was inversely correlated with lag phase (r 5 20.39, p ,0.05) suggesting an unfavorable influence on the susceptibility of LDL to oxidation. In addition, LDL cholesterol was positively correlated with TBARS at 0 hours (r 5 0.30, p 5 0.49), and at 2 hours (r 5 0.50, p ,0.01); and conjugated dienes at 0 hours (r 5 0.43, p ,0.01) and 2 hours (r 5 0.59, p ,0.0001). Total cholesterol was a significant predictor of TBARS and conjugated dienes at 2 hours (r 5 0.32, p ,0.05, r 5 0.36, p ,0.05, respectively). No other significant lipid correlates of oxidized LDL were identified. Because LDL cholesterol is a significant predictor of oxidized LDL, we have standardized measurements of oxidized LDL for lipid content. Standardization was performed by dividing indexes of oxidized LDL by LDL cholesterol plus LDL triglyceride because these contain polyunsaturated fatty acids that are oxidizable. This technique allows assessOCTOBER 1, 1997

TABLE I Characteristics of 45 Study Participants With Coronary Artery Disease Variable Sex (% men) Ethnicity (% white) Prior MI Prior CABG CAD $3 vessels* Hx of hypertension Hx of abnormal cholesterol Former smoker Family history of premature CAD Sedentary Regular wine use Multivitamin use b-blocker use Lipid-lowering drugs†

n

Percent (%)

39 43 18 23 23 22 41 24 19 8 35 10 12 24

87.0 95.6 40.0 51.1 73.5 48.9 91.1 53.3 43.2 17.8 77.8 23.3 26.7 53.3 Mean 6 SD

Age (yr) BMI (kg/m2) Heart rate (beats/min) Body fat (%) Total cholesterol (mmol/L) (mg/dl) LDL cholesterol (mmol/L) (mg/dl) HDL cholesterol (mmol/L) (mg/dl) Triglycerides (mmol/L) (mg/dl)

60.6 28.4 64.9 30.2 5.7 219.3 3.9 149.7 1.0 38.7 1.6 138.2

6 6 6 6 6 6 6 6 6 6 6 6

9 4 11 5 1 1 1 1 0 0 1 1

*Information from cardiac catheterization available on 31 subjects. † Includes HMG-CoA reductase inhibitors (n 5 17), fibric acid derivatives (n 5 5), and nicotinic acid (n 5 2). BMI 5 body mass index; CABG 5 coronary artery bypass surgery; CAD 5 coronary artery disease; Hx 5 history; MI 5 myocardial infarction.

ment of the association between each risk factor and level of oxidized LDL, adjusting for different concentrations of LDL in the samples. Significant dichotomous predictors of oxidized LDL parameters are listed in Table II. Female sex was associated with markedly elevated levels of TBARS at 2 hours compared with men (0.23 6 0.09 vs 0.138 6 0.103 mmol/mmol lipid, p ,0.05). An identical pattern existed for the association between sex and conjugated dienes with significantly higher levels observed in women compared with men at 2 hours (18.23 6 2.50 vs 10.84 6 4.83 mmol/mmol lipid, p ,0.05). Sex remained a significant predictor of unstandardized 2-hour TBARS and 2-hour conjugated dienes in multiple regression equations controlling for age and LDL cholesterol plus LDL triglyceride. Further modeling was not performed because there were only 6 female subjects. A family history of premature coronary artery disease was predictive of higher levels of 2-hour TBARS (0.19 6 0.12 mmol/mmol lipid) compared with subjects with no such history (0.12 6 0.08 mmol/mmol lipid, p ,0.05). Former tobacco users had significantly worse levels of lipid peroxides at 0 hours compared with nonsmokers (2.35 6 1.16 vs 1.61 6 1.31 mmol/mmol lipid, p ,0.05). Family history and tobacco use results were not altered by adjustment for age, sex, and LDL cholesterol plus LDL triglyceride.

Favorable indexes of oxidized LDL were observed in wine drinkers and users of multivitamins. Regular wine consumption was associated with lower levels of conjugated dienes at 0 hours compared with no regular wine use (5.40 6 3.21 vs 9.07 6 5.53 mmol/mmol lipid, p ,0.05). Multivitamin use was associated with significantly lower levels of 0-hour TBARS and 2-hour conjugated dienes compared with no multivitamin use (0.04 6 0.02 vs 0.07 6 0.04 mmol/mmol lipid, p ,0.05 and 8.52 6 3.69 vs 12.77 6 5.26 mmol/mmol lipid, p ,0.05, respectively). Both wine and multivitamin use remained significant multivariate predictors of unstandardized oxidized LDL when adjusted for age, sex, and LDL cholesterol plus LDL triglyceride. The correlation coefficients between continuous variables and oxidized LDL parameters are listed in Table III. Body mass index was inversely correlated with lag phase (r 5 20.33, p ,0.05). Percent body fat was also associated with an unfavorable LDL oxidation status, demonstrated by an inverse correlation with lag phase (r5 20.42, p ,0.01) and a positive correlation with 2 hour dienes (r 5 0.51, p ,0.01). Body fat was an independent predictor of unstandardized 2-hour dienes when adjusted for age, sex, and LDL cholesterol plus LDL triglyceride. Heart rate was inversely correlated with lag phase (r 5 20.34, p ,0.05) and positively correlated with 0- and 2-hour TBARS (r 5 0.39, p ,0.01 and r 5 0.32, p ,0.05, respectively). Heart rate retained significance in predicting lag phase and 0-hour TBARS when adjusted for age, sex, and LDL cholesterol plus LDL triglyceride in multiple linear regression equations. When b-blocker use was added to the base model, heart rate still retained significance.

DISCUSSION We identified several risk factors for increased levels of oxidized LDL in patients with ischemic heart disease. Previous studies have suggested that elevated levels of oxidized LDL occur in diabetics, smokers, and patients with cardiovascular disease.1– 4 Because our study was limited to patients with coronary artery disease, and we excluded diabetics and smokers, we were able to further refine the relation between clinical risk factors and levels of oxidized LDL. In our study, female subjects with coronary artery disease had nearly twofold higher levels of several parameters of lipid standardized oxidized LDL compared with men. Because oxidized LDL is believed to play a role in the progression of cardiovascular disease,16 this finding is consistent with the observation that women have a worse prognosis following myocardial infarction and coronary bypass surgery, have more extensive disease by angiogram, and have a lower survival following bypass surgery compared with men.17 In vitro data suggests estrogens may favorably influence levels of oxidized LDL,18 however, because of our small sample size we were unable to examine the association between hormone replacement therapy and oxidized LDL. A family history of premature coronary artery dis-

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827

TABLE II Categorical Predictors of Lipid Standardized Low-Density Lipoprotein Oxidation Parameters in Patients With Coronary Artery Disease TBARS Mean 6 SD (mmol/mmol lipid) Variable Sex Men Women Family history of cardiovascular disease Yes No Tobacco use Former Never Exercise frequency $4 3/week ,4 3/week Wine consumption Yes No Multivitamin use Yes No

Lipid Peroxides

Conjugated Dienes

Mean 6 SD (mmol/mmol lipid)

Mean 6 SD (mmol/mmol lipid)

0 Hour

2 Hour

0 Hour

2 Hour

0 Hour

2 Hour

0.06 6 0.03 0.06 6 0.04

0.14 6 0.10 0.23 6 0.09*

2.04 6 1.24 1.74 6 0.85

2.71 6 1.63 3.07 6 1.07

8.13 6 5.62 9.04 6 2.54

10.84 6 4.83 18.23 6 2.50*

0.07 6 0.04 0.05 6 0.03

0.19 6 0.12* 0.12 6 0.08

2.20 6 1.47 1.86 6 0.94

3.16 6 1.50 2.47 6 1.58

7.56 6 5.97 8.76 6 4.82

11.88 6 4.29 11.78 6 5.90

0.05 6 0.03 0.07 6 0.03

0.16 6 0.13 0.14 6 0.08

2.35 6 1.16* 1.61 6 1.31

2.81 6 1.38 2.70 6 1.79

9.79 6 6.21* 6.49 6 3.39

12.22 6 5.60 11.38 6 4.87

0.06 6 0.03 0.07 6 0.04

0.15 6 0.10 0.15 6 0.11

1.84 6 0.10 2.16 6 1.36

2.74 6 1.33 2.78 6 1.79

6.93 6 4.49 9.52 6 5.80*

11.47 6 5.83 12.16 6 4.70

0.07 6 0.04 0.06 6 0.03

0.14 6 0.09 0.15 6 0.11

1.62 6 1.12 2.11 6 1.21

2.35 6 1.75 2.88 6 1.51

5.40 6 3.21 9.07 6 5.53*

9.22 6 3.75 12.57 6 5.39

0.04 6 0.02 0.07 6 0.04*

0.10 6 0.08 0.17 6 0.11

1.82 6 0.86 2.06 6 1.28

2.44 6 0.83 2.85 6 1.72

6.51 6 4.13 8.75 6 5.55

8.52 6 3.69 12.77 6 5.26*

*p ,0.05.

TABLE III Correlation Coefficients for Continuous Variables and Lipid Standardized Low-Density Lipoprotein Oxidation Parameters in 45 Patients With Coronary Artery Disease TBARS

Lipoperoxides

Conjugated Dienes

Variable

0 Hour

2 Hour

0 Hour

2 Hour

0 Hour

2 Hour

Lag

Age BMI % Body fat Heart rate

0.09 20.05 0.06 0.39†

20.08 0.28 0.29 0.32*

20.27 0.16 20.09 0.09

20.14 0.04 0.11 20.15

20.29 0.29 0.21 20.01

20.14 0.24 0.51† 0.02

20.28 20.33* 20.42† 20.34*

*p ,0.05; †p ,0.01. BMI 5 body mass index.

ease was indicative of higher levels of TBARS. The mechanism for this may be through other inherited or shared environmental cardiovascular risk factors that may increase lipid peroxidation. We were not able to evaluate possible mechanisms in which clinical predictors may increase risk for oxidized LDL due to the limited power of multiple regression analyses. Former tobacco users had increased levels of lipid peroxides and conjugated dienes. Cigarette smoke contains high concentrations of free radicals that are capable of inducing lipid peroxidation, and smokers have been shown to have depressed levels of plasma antioxidants.19 In addition, HDL cholesterol may protect LDL from peroxidation and smokers may have decreased levels of HDL cholesterol.20 Although we did not include current smokers in our trial, a potential adverse effect of former smoking on cardiovascular disease may not be extinguished for several years. An alternative explanation may be that former smokers have other characteristics or lifestyle habits that may be associated with increased oxidized LDL compared with nonsmokers. 828

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Other clinical predictors of a less favorable LDL oxidation status included indexes of obesity. Percent body fat was positively correlated with conjugated dienes and inversely correlated with lag phase. In addition, body mass index was inversely correlated with lag phase. This is consistent with an increased risk of cardiovascular disease associated with obesity.21 It is believed that obesity may not be an independent risk factor for cardiovascular disease, but that it may increase cardiac risk through metabolic effects, such as dyslipidemia, glucose intolerance, insulin resistance and hypertension, all of which may be associated with increased oxidant stress. We were unable to explore the mechanisms through which obesity may be associated with an adverse oxidation status. A lower heart rate at rest was associated with a more favorable LDL oxidation status, including lower levels of TBARS and a longer lag phase. Previous work by Croft et al,22 demonstrated evidence for prolonged lag phase in patients with coronary disease who were taking b blockers. We considered b-blocker OCTOBER 1, 1997

use as a possible confounder in multiple regression equations because of the well-established inverse association between heart rate and use of b blockers. Heart rate remained a significant independent predictor of lag phase. A lower heart rate at rest may be associated with improved physical fitness; however, exercise level was not an independent predictor of oxidized LDL in our study. This may be due to the small variation in exercise levels observed in our subjects. Adaptation to regular exercise training may result in an improved antioxidant status.23 This observation is consistent with our univariate results. It is likely the favorable association between heart rate and LDL oxidation status we observed is related to physical condition or possibly b-blocker use; we were unable to adequately control for these factors in multiple regression analyses. Favorable levels of oxidized LDL were also observed among wine drinkers and users of multivitamins. Mean levels of conjugated dienes in regular wine users were nearly half that of nonusers. This is consistent with data that show a reduction in cardiovascular risk in subjects who consume alcohol on a regular basis24 and in particular, those who consume wine.25 Phenolic substances in red wine may inhibit the oxidation of LDL, and plasma levels of alpha-tocopherol and retinol have been shown to be elevated in red wine drinkers, suggesting an improved LDL oxidation status associated with red wine use.26 In addition, wine use was strongly correlated with HDL cholesterol in our study (r 5 0.54, p 5 0.0002), which may be protective for oxidized LDL. Multivitamin supplement use was associated with reduced TBARS and conjugated dienes. Vitamin supplements have been shown in clinical trials to reduce the oxidizability of LDL, which is also consistent with our findings.27 Furthermore, in a recent secondary prevention trial of 2,002 patients with coronary artery disease, a significant 77% reduction in nonfatal myocardial infarction was observed in participants randomized to receive either 400 or 800 IU of vitamin E compared with placebo.28 We cannot exclude the possibility that selection bias or confounding factors may account for the apparent protective effect of multivitamins on LDL oxidation in our subjects. Study limitations: Our study has several limitations. We cannot determine cause and effect due to the cross-sectional design. The purpose of this study was determine if easily identified cardiovascular risk factors could predict levels of oxidized LDL and to generate hypotheses that could be tested in studies designed to evaluate the mechanisms through which risk factors for oxidized LDL operate. Our results indicate several predictors of oxidized LDL that should be considered in evaluating the effect of interventions, such as antioxidant supplementation, on the status of LDL oxidation. We had limited power to detect differences in LDL oxidation for several dichotomous variables due to the small sample size. In particular, the number of

women included in this study was small. Our results may not be generalizable to healthy populations since our subjects had extensive cardiovascular disease. The sensitivity and specificity of the laboratory measures for LDL oxidation are limited. Laboratory imprecision tends to bias results to the null and make it more difficult to identify significant relationships. We attempted to overcome this by using multiple indexes of LDL oxidation. In a review of laboratory methodology to measure LDL oxidation, Chait29 suggested that multiple techniques be utilized for research purposes until a more sensitive and specific assay be developed. We used repeated measures of all indexes of oxidized LDL on each subject to address biologic variability and to reduce measurement error. The consistency of risk factors for oxidized LDL across outcomes, the similar findings for univariate and multivariate analyses, and the highly significant p values we obtained in conjunction with the biologic plausibility of our findings suggests that risk factors for oxidized LDL can be identified.

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