In 69 statin-treated male coronary patients with low-density lipoprotein cholesterol at goal levels (<70 mg/dl), the investigators tested whether low high-density lipoprotein (HDL) cholesterol (<40 mg/dl) and high triglyceride (>150 mg/dl) are associated with dysfunctional HDL particles and abnormal insulin, adiponectin, C-reactive protein serum levels. Thirty-four patients with low HDL cholesterol and high triglyceride (dyslipidemia) and 35 patients with low-density lipoprotein cholesterol, HDL cholesterol, and triglyceride at target levels (normolipidemia) were studied. Twenty healthy men were also studied. High-sensitivity C-reactive protein was measured using immunonephelometry, insulin using a radioimmunometric assay, and total adiponectin by enzyme-linked immunosorbent assay. Cell cholesterol efflux to serum and total isolated HDL was assayed using rat hepatoma Fu5AH cells for scavenger receptor class B type 1–mediated efflux. Compared to the normolipidemia and healthy groups, and after adjustment for age and waist circumference, patients with dyslipidemia showed higher fasting insulin (14, 9.9, and 8.5 μU/ml, respectively), homeostasis model assessment of insulin resistance values (3.4, 2.3, and 1.8, respectively), lower adiponectin concentrations (5.1, 8.1, and 11 μg/ml, respectively), and reduced cholesterol efflux to serum (14%, 15%, and 19%, respectively) and to HDL fractions (4.4%, 4.6%, and 5.6%, respectively) (p <0.05 for all variables). Multivariate analysis showed that adiponectin and apolipoprotein A1 accounted for 10.7% and 3.9%, respectively, of the variance in cholesterol efflux. In conclusion, the decreased cholesterol efflux and metabolic abnormalities found in the dyslipidemia group may contribute to the residual risk observed in the large statin trials and the higher morbidity and mortality in statin-treated coronary patients with low HDL cholesterol even when attaining low-density lipoprotein cholesterol <70 mg/dl.
The atheroprotective effect of high-density lipoprotein (HDL) particles may be mediated by their antioxidant, anti-inflammatory, and antithrombotic properties. The most important function of HDL is to promote the removal of cholesterol from the macrophages in the arterial wall and direct it to the liver for recycling or excretion. This process, known as reverse cholesterol transport, prevents arterial cholesterol accumulation, plaque destabilization, and the development of acute cardiovascular events. Cholesterol efflux from extrahepatic cells, including macrophages, an early step in reverse cholesterol transport, has recently been found to be inversely related to carotid intima-media thickness and the likelihood of angiographic coronary heart disease (CHD), independent of HDL cholesterol or apolipoprotein A1 (apoA1) levels. Although whole-serum or total isolated HDL from patients with low HDL cholesterol and high triglycerides (TG) have shown a reduced capacity to promote cell cholesterol efflux, it is unknown whether this functional abnormality is also present in statin-treated patients with CHD with the same lipid disorder. Accordingly, we hypothesized that in statin-treated male coronary patients with low-density lipoprotein (LDL) cholesterol at goal levels but with abnormal HDL cholesterol and TG levels, the serum and HDL capacity to promote cell cholesterol efflux from cultured cells is impaired. Insulin, adiponectin, and C-reactive protein levels were also evaluated.
Methods
We included 69 statin-treated male outpatients from the National Institute of Cardiology Ignacio Chavez with established CHD, defined by personal history of myocardial infarction, angioplasty, or bypass surgery, with low LDL cholesterol (<70 mg/dl). Thirty-four patients had HDL cholesterol levels <40 mg/dl and TG levels of 151 to 500 mg/dl (dyslipidemia), and 35 patients had TG and HDL cholesterol at goal levels (normolipidemia). Patients with acute cardiovascular events within the previous 3 months; congestive heart failure; body mass index >35 kg/m 2 ; myopathy or thyroid, renal, or hepatic dysfunction; and those receiving fibrates, niacin, or resins were not included in this study. The healthy volunteer group was composed of 20 subjects who attended the institute for blood donation. They had normolipidemia, had body mass index <30 kg/m 2 , did not have evidence of acute or chronic infections, and were not receiving lipid-altering drugs. The study was performed according to the Declaration of Helsinki II and was approved by our local ethics committee, and the participants gave informed consent.
Information about personal and family histories of cardiovascular risk factors and alcohol and tobacco consumption was obtained using a standardized questionnaire. In patient groups, statin dosages and concomitant medications were registered. Weight, height, waist circumference, heart rate, and blood pressure were measured, and body mass index was calculated as weight in kilograms divided by the square of height in meters. Overweight was defined as a body mass index of 25 to 29.9kg/m 2 and obesity as a body mass index >30 kg/m 2 , and central adiposity was defined as a waist circumference >90 cm. Metabolic syndrome was defined according to the National Cholesterol Education Program Adult Treatment Panel III, except for the cut point for waist circumference of 90 cm. Plasma and serum samples were collected after a 12-hour fasting period and 20 minutes of rest in a sitting position.
Plasma total cholesterol, HDL cholesterol, TG, and glucose levels were measured using standard enzymatic procedures with a Hitachi 902 analyzer (Hitachi Corporation, Tokyo Japan). Accuracy and precision were evaluated periodically by the Centers for Disease Control and Prevention (Atlanta, Georgia). LDL cholesterol was estimated. High-sensitivity C-reactive protein, apolipoprotein B, and apoA1 concentrations were measured using immunonephelometry (BN ProSpec; Dade Behring GmbH, Marburg, Germany). Intra- and interassay coefficients of variation were <6% for all analytes. Serum insulin concentrations were determined using a radioimmunometric assay (Coat-A-Count; Diagnostic Products, Los Angeles, California). Insulin resistance was estimated with the homeostasis model assessment of insulin resistance. Serum total adiponectin was determined using enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Minneapolis, MN). Intra- and interassay coefficients of variation were <10%. Cellular cholesterol efflux was determined using Fu5AH rat hepatoma cells following the procedure described by de la Llera-Moya et al, with slight modifications. Briefly, Fu5AH cells were maintained in minimal essential medium containing 5% bovine serum, 250,000 cells per well were placed on 24-well plates, and 24 hours after plating, radiolabeled cholesterol (1,2- H cholesterol; American Radiolabeled Chemicals, Inc., St. Louis, Missouri) was added (0.5 μCi/well) and cells were incubated at 37°C for 24 hours. Cells were washed and incubated for 18 hours in minimal essential medium with 0.5% bovine serum albumin. Then, cells were washed and incubated at 37°C for 4 hours with 2.0% diluted serum or isolated total HDL fractions, as estimated by phospholipids (100 μg phospholipids/mL), prepared by sequential ultracentrifugation, as previously described. Radioactivity was measured in medium and cells, and the percentage of cholesterol efflux was calculated. All determinations were made in triplicate. As an internal control, a total serum and isolated total HDL were included in each plate. Intra- and interassay coefficients of variation for this method were 4.9% and 8.1%, respectively.
Statistical comparisons were performed using SPSS for Windows version 13.0 (SPSS, Inc., Chicago, Illinois). Results are expressed as mean ± SD for continuous variables and as percentages for categorical variables. Skewed variables were transformed into natural logarithmic values. Differences in mean values among groups were analyzed using analysis of variance or analysis of covariance. Post hoc Bonferroni’s tests were conducted. Frequencies were compared using Fisher’s exact tests. Pearson’s correlation coefficients were calculated to assess relations between variables, and multivariate stepwise linear regression analysis was performed to determine the relative contributions of the variables studied to the cholesterol efflux. All p values are 2 tailed and were considered significant if <0.05.
Results
The clinical characteristics of the 3 groups studied are listed in Table 1 . The mean age in the healthy group was lower but without statistical significance. Compared with the normolipidemia group, mean values of body mass index and waist circumference and the prevalence of obesity (27% vs 7% p = 0.025) were higher in the dyslipidemia group. This latter group also had the highest fasting glucose levels. The prevalence of diabetes, hypertension, and current smoking was similar in the 2 groups of patients. Five patients with diabetes in the dyslipidemia group and 9 patients in the normolipidemia group received glucose-lowering drugs, and the other 2 patients in these groups received only diet treatment. Two subjects, 1 from each patient group, had glycosylated hemoglobin levels of 7.4% and 7.6%; the remaining patients with diabetes had glycosylated hemoglobin levels <7%. There were no differences among the patient groups with respect to previous cardiovascular events or procedures. All patients with CHD and none from the healthy group were receiving statins. The statin most commonly prescribed was simvastatin (49%), followed by atorvastatin (23%), rosuvastatin (21%), and pravastatin (6%). When comparing the 2 patient groups, atorvastatin was more commonly used in the normolipidemia group than in the dyslipidemia group (34% vs 17%, p = 0.05). No significant differences were found between statin doses or blood pressure–lowering medications. As listed in Table 2 , total cholesterol, LDL cholesterol, and non-HDL cholesterol levels adjusted for age and waist circumference were significantly lower in the 2 patient groups than in the healthy group. As expected by the study design, patients with dyslipidemia had lower HDL cholesterol and higher TG levels ( Table 2 ). This group also showed the lowest apoA1 levels and the highest total cholesterol/HDL cholesterol ratio. Fasting insulin and homeostasis model assessment of insulin resistance were also significantly higher and adiponectin levels significantly lower in patients with dyslipidemia than in the other 2 groups in unadjusted and adjusted analyses ( Table 2 ). There were no significant differences in plasma C-reactive protein among groups.
| Variable | Coronary Patients | p Value ⁎ | ||
|---|---|---|---|---|
| Dyslipidemia | Normolipidemia | Healthy Subjects | ||
| (n = 34) | (n = 35) | (n = 20) | ||
| Age (years) | 56 ± 12 | 58 ± 12 | 53 ± 8.5 | 0.209 |
| Body mass index (kg/m 2 ) | 28 ± 4.5 ‡ | 26 ± 3.6 | 26 ± 2.5 | 0.028 |
| Waist circumference (cm) | 95 ± 11 † | 90 ± 9.8 | 88 ± 6.6 | 0.034 |
| Systolic blood pressure (mm Hg) | 126 ± 24 | 124 ± 27 | 116 ± 15 | 0.393 |
| Diastolic blood pressure (mm Hg) | 79 ± 19 | 75 ± 12 | 74 ± 10 | 0.590 |
| Glucose (mg/dl) | 97 ± 14 † | 94 ± 10 | 87 ± 5.9 | 0.005 |
| Diabetes mellitus | 6 (18%) | 10 (29%) | — | 0.394 |
| Hypertension § | 23 (68%) | 25 (71%) | — | 0.797 |
| Metabolic syndrome | 31 (91%) † , ‡ | 4 (11%) † | 0 | <0.001 |
| Current smoker (≥1 cigarette/day) | 0 | 1 (3%) | — | 1.000 |
| Myocardial infarction | 31 (91%) | 33 (94%) | — | 0.673 |
| Coronary angioplasty | 18 (53%) | 17 (49%) | — | 0.811 |
| Coronary bypass | 8 (23%) | 10 (29%) | — | 0.785 |
| Medications | ||||
| Statins | 34 (100%) | 35 (100%) | — | 1.000 |
| β blockers | 25 (73%) | 27 (77%) | — | 0.785 |
| Angiotensin-converting enzyme inhibitors | 28 (82%) | 30 (86%) | — | 0.752 |
| Diuretics | 6 (18%) | 10 (29%) | — | 0.394 |
| Calcium channel blockers | 11 (32%) | 12 (34%) | — | 1.000 |
| Aspirin | 32 (94%) | 31 (89%) | — | 0.673 |
⁎ Analysis of variance for continuous variables and Fisher’s exact tests for categorical variables.
† p <0.05 versus healthy group;
‡ p <0.05 versus normolipidemia group.
§ Blood pressure ≥140/90 mm Hg or use of antihypertensive drugs.
| Variable | Coronary Patients | p Value | ||
|---|---|---|---|---|
| Dyslipidemia | Normolipidemia | Healthy Subjects | ||
| (n = 34) | (n = 35) | (n = 20) | ||
| Cholesterol (mg/dl) | ||||
| Total | 130 ± 23 ⁎ | 129 ± 21 ⁎ | 183 ± 33 | <0.001 |
| LDL | 65 ± 23 ⁎ | 65 ± 19 ⁎ | 108 ± 30 | <0.001 |
| HDL | 31 ± 4.3 ⁎ , † | 48 ± 7.1 ⁎ | 59 ± 18 | <0.001 |
| Non-HDL | 100 ± 22 ⁎ , † | 81 ± 20 ⁎ | 124 ± 31 | <0.001 |
| TG (mg/l) | 215 ± 74 ⁎ , † | 101 ± 24 | 100 ± 27 | <0.001 |
| Total cholesterol/HDL cholesterol ratio | 4.3 ± 0.9 ⁎ , † | 2.7 ± 0.5 ⁎ | 3.2 ± 0.8 | <0.001 |
| Apolipoprotein B (mg/dl) | 76 ± 19 | 66 ± 17 ⁎ | 82 ± 17 | 0.006 |
| ApoA1 (mg/dl) | 144 ± 18 ⁎ , † | 166 ± 23 | 167 ± 38 | 0.001 |
| Insulin (μU/ml) | 14 ± 8.2 ⁎ , † | 9.9 ± 7.2 | 8.5 ± 5.6 | 0.004 |
| Homeostatic model assessment of insulin resistance | 3.4 ± 2.1 ⁎ , † | 2.3 ± 1.7 ⁎ | 1.8 ± 1.1 | 0.001 |
| Adiponectin (μg/ml) | 5.1 ± 4.4 ⁎ , † | 8.1 ± 4.9 | 11 ± 5.2 | <0.001 |
| C-reactive protein (mg/l) | 2.5 ± 3.4 | 2.1 ± 3.3 | 1.2 ± 0.8 | 0.207 |
| Cholesterol efflux to serum (%) | 14 ± 1.2 ⁎ | 15 ± 1.2 ⁎ | 19 ± 1.2 | <0.001 |
| Cholesterol efflux to total isolated HDL cholesterol | 4.4 ± 1.3 ⁎ | 4.6 ± 1.3 | 5.6 ± 1.4 | 0.041 |
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