Low-Density Lipoprotein Size, Pravastatin Treatment, and Coronary Events FREE

Small-size low-density lipoprotein (LDL) cholesterol has been associated with coronary disease in several retrospective case-control studies,^1- 6 although other studies have not found this association.^7- 10 In fact, in 2 studies, larger LDL particles were associated with increased risk of coronary disease.^7- 8 Most prospective studies,^11- 13 but not all,¹⁴ also found that patients with coronary disease are more likely than controls to have smaller LDL particles. However, no study, retrospective or prospective, found that small LDL had independent predictive value after adjustment for standard lipid risk factors.^{1- 6,11- 13}

One of the difficulties in establishing whether LDL size is an independent predictor of coronary disease or is merely a marker of other lipid abnormalities is that the predominance of small LDL is strongly associated with increased triglyceride and reduced HDL cholesterol concentrations as well as other traits of the metabolic syndrome.^15- 16 Predominance of small LDL in patients with coronary disease may simply reflect these other traits, and so its clinical utility has been questioned.^16- 17 Indeed, in studies in which triglyceride concentrations were similar in cases and controls, small LDL was either unrelated^9- 10,14 or inversely related^7- 8 with risk of coronary disease. Because of such strong confounding, the true direct relationship, if any, between LDL size and coronary disease has been difficult to determine.

The current study uses a prospective, nested case-control design to examine whether LDL size is an independent predictor of recurrent coronary events in survivors of myocardial infarction (MI) in the Cholesterol and Recurrent Events (CARE) trial. This study is unique because it is the largest prospective study of LDL size and coronary disease to date, and because the lipid levels are typical of patients with coronary disease.

The CARE trial (conducted in the period 1989-1996) was a randomized, placebo-controlled trial of pravastatin in 4159 patients who experienced acute MI 3 to 20 months before enrollment.¹⁸ The eligibility ranges for plasma lipid concentrations were typical of patients with coronary disease: total cholesterol, less than 240 mg/dL (6.22 mmol/L); LDL cholesterol, 115 to 174 mg/dL (2.98-4.51 mmol/L); and triglycerides, less than 350 mg/dL (3.96 mmol/L). The median duration of follow-up was 5 years. Fasting venous blood was taken from each patient on each of 2 screening visits, at least 1 week apart, and sent by overnight delivery in cooled containers to the core laboratory in St Louis, Mo. Plasma was separated in a refrigerated centrifuge and 1-mL aliquots were placed in polypropylene vials and stored at –80°C until analysis. Cases were those patients who experienced a primary end point (coronary death or confirmed MI) during the follow-up period (n = 486).¹⁸ Patients who did not experience a primary end point were randomly selected and matched to cases by decade of age (eg, 40-49 years, 50-59 years) and by sex. Sufficient plasma from 2 screening visits was available for LDL size analysis in 416 cases and 421 controls.

At the conclusion of the 5-year follow-up period, vials containing frozen plasma from the 2 screening visits were shipped by overnight delivery to the laboratory at the Harvard School of Public Health for determination of LDL size. The LDL peak diameter was determined from whole plasma by lipid-stained, nondenaturing gel electrophoresis using 2% to 16% polyacrylamide gradient gels.^19- 20 Gels were stained for lipid with Sudan black (Sigma, St Louis, Mo) and scanned with a laser densitometer (LKB-Ultroscan LX, LKB Instruments Inc, Paramus, NJ). Size of LDL was estimated from calibration curves that were constructed using latex beads (Duke Scientific Corp, Palo Alto, Calif) and high molecular-weight standards (Pharmacia AB, Stockholm, Sweden) as previously described.¹⁹ To ensure accuracy in the determination of LDL size, our values were standardized by sending control samples to Ronald Krauss, MD, at the Donner Laboratory, Berkeley, Calif. These control samples had LDL diameters smaller than 25.5 nm, between 26.0 and 26.5 nm, and larger than 27.0 nm. The deviation between our laboratory and the Donner Laboratory was less than 0.25% for all samples. Subjects with a predominant LDL peak size of 25.5 nm or smaller were classified as "pattern B," and those with LDL size larger than 25.5 nm were "pattern A."²¹ Matched cases and controls were included together in 1 gel. Laboratory personnel were blinded to case-control status. The between-run coefficient of variation was 0.95% for the internal control sample included in every gel, and 0.54% for 20 blinded duplicate samples interspersed throughout the samples in the study. Plasma triglyceride, LDL cholesterol, and high-density lipoprotein (HDL) cholesterol concentrations were measured as previously described.²²

Statistical analyses were performed at the University of Texas School of Public Health, Houston, using SAS version 8.1 (SAS Institute Inc, Cary, NC). The distribution of LDL size of the controls was used to compute quintiles and the number of cases and controls in each quintile was then determined. These quintile categories were also used in subgroup analyses, and therefore quintiles from controls in subgroup analyses were not evenly distributed. Multiple logistic regression computed relative risks (RRs) with 95% confidence intervals (CIs) for case status for the second through fifth quintiles compared to the first quintile. Tests for linear trend were performed on the RRs across quintiles, using the median value for each of the quintiles. The univariate model included age only, while the basic multivariable models used for all analyses included age, smoking, hypertension, and left ventricular ejection fraction.

Other covariates that were associated with LDL size as well as with coronary events, and thus could be part of a causal pathway between LDL size and coronary events, were studied in additional models. These covariates were use of β-adrenergic antagonists, use of diuretics, waist circumference, history of diabetes, and the standard lipid risk factors LDL cholesterol, HDL cholesterol, and triglycerides. Waist circumference was the covariate used to account for obesity since it correlated with LDL size and predicted recurrent coronary events in this population (4% increase in risk of coronary events per 2.5 cm of waist circumference, P = .03),²² and because it is now recommended to use in clinical practice.²³

The protocol specified analyses in the total group regardless of assignment to placebo or pravastatin, as well as in each treatment group separately. Tests for interaction between treatment assignment, LDL size, and RR of coronary events were conducted. The cutoff for statistical significance was P = .05 (2-sided). There were 460 patients in the placebo group and 377 patients in the pravastatin group.

Because of the potential effects of β-blockers on LDL size,⁴ we repeated the analysis after excluding all those patients who were taking β-blockers at baseline. In the placebo group, there were 129 cases and 132 controls not taking β-blockers.

Previous studies carried out in this population²² excluded control patients who had had coronary artery bypass graft (CABG) surgery, coronary angioplasty, or stroke after randomization during the follow-up. These subjects were excluded because results from the trial showed that pravastatin reduced coronary revascularization and stroke,¹⁸ and patients with these clinical end points may not be considered event-free controls. To evaluate whether the inclusion of these subjects in our study could have affected the results, we also repeated the analysis in the placebo group including only those controls who had no history of CABG surgery, coronary angioplasty, or stroke after randomization during the follow-up.

The characteristics of cases and controls are shown in Table 1. The mean LDL size was identical in cases and controls, 25.6 nm. The prevalence of pattern B, denoting a predominance of small LDL, was 39% in cases and 40% in controls. Patients in the highest quintile of LDL size were older, less obese, had lower triglyceride and total cholesterol concentrations, and higher HDL cholesterol concentrations (Table 2). The prevalence of hypertension and diabetes, blood pressure levels, and medication use was not significantly associated with LDL size. LDL size was significantly (P<.01) correlated with triglycerides (r = −0.63), HDL cholesterol (r = 0.50), and LDL cholesterol (r = 0.13).

Analysis of the total cohort (those randomized to placebo or pravastatin) showed that LDL size was not a significant predictor of recurrent coronary events in a multivariable model that included age, smoking, history of hypertension, and left ventricular ejection fraction (RR, 1.27; 95% CI, 0.82-1.98; P = .28 for the highest quintile compared with the lowest). When diabetes, waist circumference, and use of β-adrenergic antagonists and diuretics were added to the model, large LDL was a significant independent predictor of coronary events (RR, 1.60; 95% CI, 1.00-2.55; P = .049). Further adjustment for plasma triglycerides, LDL cholesterol, and HDL cholesterol strengthened this association (adjusted RR, 2.10; 95% CI, 1.20-3.68; P = .01).

The relationship between large LDL size and coronary events was evident only in the patients in the placebo group (P = .046 for a difference in the effect of LDL size on coronary events between the placebo and treatment groups). Thus the results are presented separately for each treatment group. Large LDL was a significant predictor of recurrent coronary events in the univariate model that only included age as a covariate (Table 3). When smoking, history of hypertension, left ventricular ejection fraction, use of β-adrenergic antagonists and diuretics, diabetes, and waist circumference were added to the model, the risk for recurrent coronary events was increased. Further adjustment for plasma triglycerides, LDL cholesterol, and HDL cholesterol considerably strengthened the association between large LDL and the risk of recurrent coronary events (Table 3 and Figure 1).

Size of LDL was not a predictor of recurrent events in the pravastatin group. In the age-adjusted model, the RR of a recurrent event for the highest quintile of LDL size was 0.98 (95% CI, 0.47-2.04; P = .88 for trend). In a multivariable model adjusted for lipid and nonlipid risk factors, the RR was only 1.33 (95% CI, 0.52-3.38) (for trend, P = .56; for interaction, P = .11) (Figure 1).

Exclusion of patients who were taking β-blockers did not affect the findings. In the placebo group, large LDL size was a predictor of recurrent coronary events in the univariate model that only included age as a covariate (RR, 2.15; 95% CI, 0.98-4.76; P = .06 for the highest compared with the lowest quintile). In the multivariable model that included all the nonlipid risk factors the RR was 2.44 (95% CI, 1.05-5.68; P = .04). Adding triglyceride, LDL cholesterol, and HDL cholesterol to the model further strengthened the association (RR, 4.22; 95% CI, 1.50-11.92; P = .007).

Similarly, excluding control patients who had had CABG surgery, coronary angioplasty, or stroke after randomization during the follow-up did not affect the results. There was a positive trend in the model that included the covariates age, smoking, history of hypertension, and left ventricular ejection fraction (RR, 1.77; 95% CI, 0.98-3.22; P = .10 for trend), which became statistically significant when diabetes, waist circumference, and the use of β-adrenergic antagonists and diuretics were added to the model (RR, 2.14; 95% CI, 1.15-3.98; P = .03 for trend). After adding plasma triglycerides, LDL cholesterol, and HDL cholesterol to the model, the association between large LDL and the risk of recurrent coronary events was further increased (RR, 3.66; 95% CI, 1.67-8.02; P = .003 for trend).

We found that large LDL size was a significant predictor of increased recurrent coronary events in a typical population of survivors of MI. This association was robust: it was present in both univariate and multivariable analyses and was independent of other plasma lipid and nonlipid risk factors. Increased LDL size was a risk factor among patients in the placebo group (4-fold increase) but not in the pravastatin group. These findings are contrary to the prevailing view that a predominance of small LDL predicts coronary disease events.^15,21

The association between large LDL and coronary disease has been previously described. Large LDL was independently associated with increased risk of coronary disease in Canadian normolipidemic patients.⁷ Among American Indian communities, large LDL was associated with higher coronary disease mortality.⁸ Cross-cultural studies also suggest that large LDL is associated with clinical coronary disease. In Finland, where coronary disease incidence is very high, the mean LDL size is very large (27.1 nm) and the prevalence of pattern B, the small-LDL phenotype, is very low (15%).¹⁴ In contrast, in a healthy rural Costa Rican population with a low prevalence of coronary disease, LDL size is smaller (mean, 26.2 nm) and the prevalence of pattern B higher (44%) than in Finland.²⁴

Because of the focus on small LDL size as a marker of coronary disease, evidence that large LDL size is atherogenic has not been well recognized. Large LDL particles, similar in size to those found in humans, predominate in hypercholesterolemic pigs that are more susceptible to atherosclerosis.²⁵ In nonhuman primates, a diet high in saturated fat and cholesterol increases LDL size, and the magnitude of this increase is strongly associated with severity of atherosclerosis.²⁶ In humans as well, diets that are high in saturated fat and cholesterol increase the plasma concentration of large LDL.²⁷

Large LDL particles are thought to be large because of high cholesterol ester content. They preferentially bind to isolated arterial proteoglycans,²⁸ delivering more cholesterol per particle to cells and connective tissue in the arterial wall.^26,29 Taken together, evidence from epidemiologic and atherosclerosis studies suggests that large cholesterol-ester–rich LDLs are atherogenic and predictive of coronary disease events in humans. Small LDL does have potentially deleterious properties such as reduced affinity for the LDL receptor,³⁰ longer residence time in plasma,³¹ increased susceptibility to oxidation,³² and adverse effects on the function of vascular cells.¹⁵ However, these may not be worse, in vivo, than the harmful properties of large LDL.

In our study, large LDL size did not predict coronary events in the pravastatin group. This may be because pravastatin reduces the concentration of larger LDL particles, as suggested by a major decrease in average LDL size (−7 nm, P = .01) and reduced cholesterol ester content of LDL during pravastatin therapy.³³

We investigated reasons why previous studies found that small LDL is associated with coronary disease. One difference is that the present study pertained to recurrent events in patients after MI, while previous prospective studies were in populations that had not experienced a major coronary event at baseline. However, there has been no difference in how the lipid risk factors predict initial or recurrent events.^18,34- 35 In previous studies, small LDL was associated with coronary disease in univariate analyses that did not include the well-established lipoprotein risk factors. In prospective studies, small LDL has not remained a significant independent predictor of coronary disease after adjustment for total cholesterol and triglycerides,¹² ratio of total cholesterol to HDL cholesterol,¹¹ or apolipoprotein B,¹³ all of which were significant predictors despite the inclusion of LDL size in the model. Similarly, retrospective case-control studies did not find that small LDL size remained significantly associated with coronary disease in multivariate analysis.^1- 2,4- 5 These findings suggest that confounding by other lipid risk factors accounts for the association of small LDL with coronary disease.

Comparison of 1 such study, the Physicians' Health Study (PHS),¹² with CARE provides insight on the importance of confounding. In PHS, there was a difference of 53 mg/dL (0.60 mmol/L) in triglyceride concentrations between MI cases and controls compared with CARE, where this difference was only 9 mg/dL (0.10 mmol/L). In PHS, cases had significantly smaller LDL compared with controls, whereas in CARE the mean LDL size was identical in cases and controls. Thus, confounding between high triglycerides and small LDL was present in PHS, much more so than in CARE.

In both studies, triglyceride was an independent risk factor. Triglyceride metabolism is thought to play an important role in determining structure and composition of LDL,³⁶ with small LDL a byproduct of hypertriglyceridemia.³⁷ In CARE, the confounding influence of triglyceride with LDL size was less than in PHS, permitting a true relationship to emerge between large LDL size and coronary disease. Other studies failed to detect a significant independent association between large LDL size and coronary disease, perhaps because of residual confounding by such highly correlated lipid risk factors. Taken together, these data support the hypothesis that attenuation of the association between LDL size and other lipids reveals an independent relationship between large LDL and coronary disease.

One limitation of the study presented herein is that LDL size was not measured during the follow-up period. Also, while it is unlikely that medications other than β-blockers and diuretics may have had an effect on LDL size, the possibility cannot be completely excluded.

In conclusion, large LDL size was an independent predictor of coronary events in a population typical of patients with cardiovascular disease. This adverse effect was not present among patients who were treated with pravastatin. Identifying patients on the basis of LDL size may not be useful clinically, since effective treatment for elevated LDL cholesterol concentrations also effectively treats risk associated with large LDL.