Abstract
Objective
A direct assay for small dense low density lipoprotein cholesterol (sdLDL-C) has been developed. Our goal was to establish normal ranges for this assay as well as to measure values in patients with established coronary heart disease (CHD) versus control subjects.
Methods
Direct LDL-C and sdLDL-C analyses were carried out on samples from 3,188 male and female participants of the Framingham Offspring Study, which included 173 male and 74 female CHD cases.
Results
Male gender and female postmenopausal status were both associated with significantly (p<0.0001) higher sdLDL-C values. Use of cholesterol-lowering medications was significantly (p<0.0001) higher in CHD cases than in controls (46.8% versus 11.4% in men, and 35.1% versus 8.8% in women). Direct LDL-C levels were significantly lower in male CHD patients than in male controls (3.22 versus 3.51 mmol/L, p<0.0001), but their mean sdLDL-C levels were similar to those in controls (0.83 versus 0.84 mmol/L, p=0.609). Female CHD patients had similar LDL-C values to female controls (3.53 versus 3.46 mmol/L, p=0.543), but had significantly higher sdLDL-C values (0.83 versus 0.68 mmol/L, p=0.0015). Both male and female cases also had significantly (p<0.01) higher percentages of LDL-C as sdLDLC than controls.
Conclusions
Despite four fold greater cholesterol lowering therapy use, CHD patients had mean LDL-C values well above the LDL-C goal of < 2.6 mmol/L or 100 mg/dl, and male CHD cases had similar sdLDL C values and female CHD cases had significantly higher values than controls. These findings may explain some of the high residual risk of future CHD events in CHD patients.
Keywords: small dense LDL-cholesterol, coronary heart disease, risk factor, Framingham Offspring Study, obesity
Introduction
Elevated plasma low-density lipoprotein cholesterol (LDL-C) levels have been shown to be a significant risk factor for coronary heart disease (CHD), and lowering LDL-C with medications has been shown to reduce heart disease risk (1,2). LDL is comprised of a variety of different subfractions, which can be separated by ultracentrifugation, gradient gel electrophoresis, nuclear magnetic resonance, and specific precipitation methods (3-7). Gradient-gel electrophoresis and nuclear magnetic resonance imaging are semi-quantitative methods. For nuclear magnetic resonance analysis a significant number of assumptions are required to estimate concentrations of LDL subfractions as well as total LDL particle number (8,9). Hirano and colleagues have developed a methodology for measuring small dense LDL (sdLDL) cholesterol directly that correlates highly with the measurement of cholesterol in sdLDL at a density between 1.044 – 1.063 g/ml (10-13). Our goal was to use this assay along with direct lipoprotein cholesterol levels in participants in cycle 6 of the Framingham Offspring Study in order to develop normal ranges for this new assay, as well as to determine potential differences in CHD cases versus controls.
Methods
Study Subjects
Participants in the Framingham Offspring Study (FOS), a long-term community-based prospective observational study of risk factors for CHD are the offspring and their spouses of the original Framingham Heart Study (FHS) cohort (14-17). During cycle 6 of FOS (1995–1998) participants underwent a standardized medical history, physical examination, and fasting lipid measurements. All samples were stored in our laboratory at -80 °C, and never thawed until use. Selection criteria for the CHD cases included a history of myocardial infarction, acute coronary insufficiency, or angina pectoris at cycle 6. None of the subjects had acute coronary syndrome at the time of the examination. We performed our analyses on all available plasma samples from male and female participants in cycle 6. To determine a normal range for sdLDL-C, we selected male and female participants of FOS (cycle 6) without CHD or diabetes and not taking cholesterol-lowering medications or hormonal replacement therapy (1080 men and 1012 women). We determined gender differences in the normal population. In normal women, we also determined differences between premenopausal (n = 313) and postmenopausal (n=698) women. We also determined differences between CHD cases and controls (subjects with no evidence of CHD at cycle 6) in each gender. There were 173 male CHD cases and 1335 male controls, and 74 female cases and 1606 female controls. We did not exclude patients on cholesterol lowering medication from this analysis.
Laboratory Measurements
Total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined by standard enzymatic methods as previously described. HDL cholesterol was measured after isolation of the HDL supernatant following dextran sulfate magnesium precipitation (18). LDL-C was calculated using the Friedewald formula (19). For the purposes of this study, archived plasma samples frozen at -80° and never thawed, were used for the assessment of direct LDL-C and sdLDL-C by automated standardized enzymatic analysis on a Hitachi 911 automated analyzer using kits provided by the Denka Seiken Corporation, Tokyo, Japan. Within- and between-run coefficients of variation for the direct LDL-C assay were 0.77% and 1.30%, while for sdLDL-C these values were 4.99% and 4.67% respectively. Large LDL-C values were calculates as: direct LDL-C – sdLDL-C, and we also calculated the percentage of LDL-C as sdLDL-C based on direct measurements.
Assays for direct LDL and sdLDL cholesterol have previously been calibrated and directly compared with values obtained after isolation of LDL and sdLDL by ultracentrifugation, and very similar results were obtained (10). For the sdLDL-C assay, plasma (0.1 ml) was combined with a precipitation reagent (0.1 ml) containing heparin sodium salt and MgCl2, and incubated for 10 minutes at 37°C. Then sdLDL (d = 1.044-1.063 g/ml) and HDL were collected by filtering off the more buoyant lipoproteins, after which sdLDL-C was measured by the homogenous method (10-13). When we compared values obtained for direct LDL-C and sdLDL-C in fresh plasma (n=20) versus values obtained in plasma stored at -80°C for 3 months, virtually identical results were obtained. All laboratory personnel were blinded with regard to the clinical status of study subjects.
Statistical Analysis
Descriptive statistics, means ± standard deviations (SD) for continuous variables or proportions for categorical variables, were computed for all study variables and all study groups. The distribution of the variables was compared between subjects with or without prevalent CHD, using two sample t tests (log-transformed, if necessary) for continuous variables and chi-square tests for categorical variables.
Results
In Table 1 we provide data on male and female participants of FOS (cycle 6) without CHD or diabetes and not taking cholesterol lowering medications or hormonal replacement therapy. Men and women had similar ages; however, body mass index, waist circumference, systolic and diastolic blood pressure, and prevalence of use of antihypertensive treatment, aspirin, or drinking more than one alcoholic beverage per week were all significantly higher (p<0.0001) in men than in women. The percentage of cigarette smokers was similar between men and women. With regard to lipid parameters, women had significantly (p<0.0001) higher levels of total cholesterol and HDL-C than men, while men had significantly (p<0.0001) higher levels of triglyceride and higher total cholesterol/HDL-C ratios than did females. With regard to non-HDL-C and LDL-C, men and women had similar values, but men had significantly (p<0.0001) higher sdLDL-C and higher percentages of LDL as sdLDL-C than women. Men were less likely to have an LDL-C level in the optimal range of less than 2.6 mmol/L (100 mg/dl) as defined by the National Cholesterol Education Program (20). Men were also much less likely to have sdLDL-C levels less than 0.5 mmol/L (20 mg/dl) than women. This value was based on approximate 25th percentile values in control subjects. Moreover, men were much more likely to have elevated sdLDL-C values in excess of 1.0 mmol/L (40 mg/dl) than women. This value was based on approximate 75th percentile values in the control male population. Men were also significantly more likely to have elevated total triglyceride values above 1.7 mmol/L (150 mg/dl) and less likely to have non-HDL cholesterol levels of less than 3.4 mmol/L (130 mg/dl). These latter cutpoints were identified by the National Cholesterol Education Program Adult Treatment Panel III (20).
Table 1.
Characteristics, disease prevalence, and plasma lipid levels in men and women participating in the Framingham Offspring Study (cycle 6) without coronary heart disease, diabetes mellitus, cholesterol-lowering medications and hormone replacement therapies
| Variable | Men (n = 1080) |
Women (n = 1012) |
p value |
|---|---|---|---|
|
|
|||
| Age (years) | 57.1 ± 9.7 | 57.4 ± 10.5 | 0.4851 |
| Body Mass Index (kg/m2) | 28.2 ± 4.3 | 27.1 ± 5.6 | <0.0001 |
| Body Mass Index > 30 (%) | 27.2 | 24.2 | * 0.1146 |
| Waist (cm) | 100.4 ±10.7 | 93.1 ± 14.7 | <0.0001 |
| Waist > 102cm (%) | 39.8 | 23.6 | * <0.0001 |
| Systolic Blood Pressure (mmHg) | 128.3 ± 16.5 | 124.8 ± 19.3 | <0.0001 |
| Diastolic Blood Pressure (mmHg) | 77.8 ± 9.3 | 73.7 ± 9.3 | <0.0001 |
| Hypertension (%) | 36.1 | 30.7 | * 0.0091 |
| Hypertensive Treatment (%) | 21.2 | 17.9 | * 0.0561 |
| Taking Aspirin Regularly (%) | 26.1 | 17.6 | * <0.0001 |
| Cigarette Smokers (%) | 15.1 | 16.2 | * 0.4832 |
| > One drink alcohol/week (%) | 55.9 | 30.9 | * <0.0001 |
| TC (mmol/l) | 5.21 ± 0.92 | 5.47 ± 1.02 | <0.0001 |
| TG (mmol/l) | 1.29 [0.91,1.81] | 1.13 [0.82, 1.60] | #<0.0001 |
| HDL-cholesterol (mmol/l) | 1.16 ± 0.32 | 1.48 ± 0.39 | <0.0001 |
| TC/HDLC ratio | 4.66 [3.75, 5.63] | 3.69 [3.01, 4.67] | #<0.0001 |
| non-HDL-cholesterol (mmol/l) | 4.05 ± 0.93 | 3.99 ± 1.06 | 0.2268 |
| calculated LDL-cholesterol (mmol/l) | 3.38 ± 0.82 | 3.39 ± 0.92 | 0.7218 |
| direct LDL-cholesterol (mmol/l) | 3.54 ± 0.84 | 3.49 ± 0.94 | 0.1540 |
| small dense LDL-cholesterol (mmol/l) | 0.82 ± 0.39 | 0.67 ± 0.41 | <0.0001 |
| % of LDL as sdLDL cholesterol | 22.9 ± 8.9 | 18.6 ± 8.7 | <0.0001 |
| large LDL-cholesterol (mmol/l) | 2.72 ± 0.69 | 2.82 ± 0.75 | 0.0013 |
| Fasting Glucose (mmol/l) | 5.51 ± 0.51 | 5.28 ± 0.56 | <0.0001 |
| LDL-cholesterol < 2.6mmol/l (%) | 12.2 | 16.9 | * 0.0024 |
| LDL-cholesterol > 4.2mmol/l (%) | 23.8 | 22.7 | * 0.5631 |
| sdLDL-C < 0.5mmol/l (%) | 24.1 | 43.5 | * <0.0001 |
| sdLDL-C > 1.0mmol/l (%) | 26.5 | 13.6 | * <0.0001 |
| Triglyceride > 1.7mmol/l (%) | 28.5 | 22.1 | * 0.0008 |
| nonHDL-C < 3.4mmol/l (%) | 22.2 | 27.4 | * 0.0061 |
| nonHDL-C > 4.9mmol/l (%) | 16.3 | 18.6 | * 0.1651 |
Values are expressed as mean ± SD, median [25th percentile, 75th percentile], or percentage.
p-value obtained by Chi-Square test
p-value obtained after log-transformation of the data.
In Table 2 we provide information on the differences between premenopausal and postmenopausal women. Postmenopausal women had significantly (p<0.0001) greater waist circumferences, higher systolic blood pressure levels, were more likely to have a history of hypertension, to be on antihypertensive therapy as well as to be taking aspirin than premenopausal women. Postmenopausal women had significantly (p<0.0001) higher total cholesterol, triglyceride, total cholesterol/HDL cholesterol ratio, non-HDL cholesterol, calculated LDL-C, direct LDL-C, and sdLDL-C values than did premenopausal women. They also had significantly higher large LDL-C and higher fasting glucose values.
Table 2.
Characteristics, disease prevalence, and plasma lipid levels in premenopausal and postmenopausal women participating in the Framingham Offspring Study (cycle 6) without coronary heart disease, diabetes mellitus, cholesterol-lowering medications and hormone replacement therapies
| Variable | Premenopausal (n = 313) |
Postmenopausal (n = 698) |
p value |
|---|---|---|---|
|
|
|||
| Age (years) | 46.4 ± 5.0 | 62.4 ± 8.3 | <0.0001 |
| Body Mass Index (kg/m2) | 26.7 ± 5.5 | 27.2 ± 5.6 | 0.1622 |
| Body Mass Index > 30 (%) | 23.8 | 24.4 | * 0.8383 |
| Waist (cm) | 89.4 ± 13.6 | 94.7 ± 14.8 | <0.0001 |
| Waist > 102 cm (%) | 16.6 | 26.7 | * 0.0005 |
| Systolic Blood Pressure (mmHg) | 115.3 ± 15.5 | 129.1 ± 19.3 | <0.0001 |
| Diastolic Blood Pressure (mmHg) | 72.9 ± 9.2 | 74.0 ± 9.3 | 0.0787 |
| Hypertension (%) | 15.1 | 37.7 | * <0.0001 |
| Hypertensive Treatment (%) | 9.0 | 21.9 | * <0.0001 |
| Taking Aspirin Regularly (%) | 9.3 | 21.4 | * <0.0001 |
| Cigarette Smokers (%) | 16.3 | 16.2 | * 0.9741 |
| > One drink alcohol/week (%) | 34.8 | 29.1 | * 0.0715 |
| TC (mmol/l) | 5.03 ± 0.94 | 5.68 ± 0.99 | <0.0001 |
| TG (mmol/l) | 1.07 [0.72, 1.35] | 1.20 [0.89, 1.73] | #<0.0001 |
| HDL-cholesterol (mmol/l) | 1.47 ± 0.39 | 1.48 ± 0.39 | 0.6906 |
| TC/HDLC ratio | 3.37 [2.81, 4.20] | 3.89 [3.16, 4.86] | #<0.0001 |
| non-HDL-cholesterol (mmol/l) | 3.56 ± 1.01 | 4.19 ± 1.03 | <0.0001 |
| calculated LDL-cholesterol (mmol/l) | 3.05 ± 0.88 | 3.55 ± 0.89 | <0.0001 |
| direct LDL-cholesterol (mmol/l) | 3.17 ± 0.91 | 3.63 ± 0.93 | <0.0001 |
| small dense LDL-cholesterol (mmol/l) | 0.55 ± 0.38 | 0.72 ± 0.41 | <0.0001 |
| % of LDL as sdLDLC | 17.0 ± 9.1 | 19.3 ± 8.4 | <0.0001 |
| large LDL-cholesterol (mmol/l) | 2.62 ± 0.73 | 2.92 ± 0.74 | <0.0001 |
| Fasting Glucose (mmol/l) | 5.09 ± 0.46 | 5.37 ± 0.58 | <0.0001 |
| LDL-cholesterol < 2.6mmol/l (%) | 28.1 | 11.9 | * <0.0001 |
| LDL-cholesterol > 4.2mmol/l (%) | 12.5 | 27.4 | * <0.0001 |
| sdLDL-C < 0.5mmol/l (%) | 60.7 | 35.9 | * <0.0001 |
| sdLDL-C > 1.0mmol/l (%) | 8.6 | 15.6 | * 0.0025 |
| Triglyceride > 1.7mmol/l (%) | 12.5 | 26.5 | * <0.0001 |
| nonHDL-C < 3.4mmol/l (%) | 44.7 | 19.7 | * <0.0001 |
| nonHDL-C > 4.9mmol/l (%) | 7.1 | 23.8 | * <0.0001 |
Values are expressed as mean ± SD, median [25th percentile, 75th percentile], or percentage.
p-value obtained by Chi-Square test
p-value obtained after log-transformation of the data.
In table 3 selected percentile values for direct LDL-C, calculated LDL-C, and sdLDL-C, for normal men and women are provided. The Adult Treatment Panel of the National Cholesterol Education Program has selected approximate 75th percentile values (4.15 mmol/L or 160 mg/dl) for LDL-C as being associated with high CHD risk. The 75th percentile for LDL-C in men was 4.10 mmol/L and in women this value was 4.08 mmol/L, with somewhat lower values for calculated LDL-C. The 75th percentile sdLDL-C value was 1.05 mmol/L (40 mg/dl) for men, 0.91 mmol/L (35 mg/dl) for postmenopausal women and 0.65 mmol/L (25 mg/dl) for premenopausal women.
Table 3.
Means and selected percentiles of direct LDL-C, calculated LDL-C, small dense LDL-C, large LDL-C, and sdLDLC/LDLC ratio in participants of the Framingham Offspring Study (cycle 6) without coronary heart disease, diabetes mellitus, cholesterol-lowering medications and hormone replacement therapies, by gender and menopausal status for women
| n | Mean ± SD | Percentiles
|
|||||
|---|---|---|---|---|---|---|---|
| 10 | 25 | 50 | 75 | 90 | |||
| direct LDL-cholesterol (mmol/l) | |||||||
| Men | 1080 | 3.54 ± 0.84 | 2.49 | 2.96 | 3.51 | 4.10 | 4.67 |
| Women | 1012 | 3.49 ± 0.94 | 2.32 | 2.85 | 3.41 | 4.08 | 4.74 |
| Premenopausal | 313 | 3.17 ± 0.91 | 2.15 | 2.47 | 3.04 | 3.74 | 4.24 |
| Postmenopausal | 698 | 3.63 ± 0.92 | 2.51 | 3.00 | 3.50 | 4.22 | 4.86 |
| calculated LDL-cholesterol (mmol/l) | |||||||
| Men | 1074 | 3.38 ± 0.82 | 2.35 | 2.81 | 3.33 | 3.93 | 4.43 |
| Women | 1008 | 3.39 ± 0.92 | 2.29 | 2.78 | 3.32 | 3.90 | 4.63 |
| Premenopausal | 313 | 3.04 ± 0.88 | 2.08 | 2.41 | 2.99 | 3.54 | 4.10 |
| Postmenopausal | 694 | 3.55 ± 0.89 | 2.44 | 2.98 | 3.47 | 4.08 | 4.72 |
| small dense LDL-cholesterol (mmol/l) | |||||||
| Men | 1080 | 0.82 ± 0.39 | 0.36 | 0.53 | 0.75 | 1.05 | 1.36 |
| Women | 1011 | 0.67 ± 0.41 | 0.27 | 0.38 | 0.57 | 0.86 | 1.17 |
| Premenopausal | 313 | 0.55 ± 0.38 | 0.23 | 0.32 | 0.45 | 0.65 | 0.99 |
| Postmenopausal | 697 | 0.72 ± 0.41 | 0.30 | 0.42 | 0.64 | 0.91 | 1.21 |
| large LDL-cholesterol (mmol/l) | |||||||
| Men | 1080 | 2.72 ± 0.69 | 1.89 | 2.24 | 2.70 | 3.14 | 3.56 |
| Women | 1011 | 2.83 ± 0.75 | 1.90 | 2.34 | 2.75 | 3.32 | 3.82 |
| Premenopausal | 313 | 2.62 ± 0.73 | 1.76 | 2.06 | 2.59 | 3.11 | 3.51 |
| Postmenopausal | 697 | 2.92 ± 0.74 | 1.98 | 2.44 | 2.83 | 3.40 | 3.92 |
| % of LDLC as sdLDLC | |||||||
| Men | 1080 | 22.9 ± 9.0 | 12.4 | 16.3 | 21.8 | 28.4 | 35.1 |
| Women | 1011 | 18.6 ± 8.7 | 9.3 | 12.7 | 16.8 | 23.2 | 29.5 |
| Premenopausal | 313 | 17.0 ± 9.1 | 8.7 | 11.5 | 14.6 | 20.7 | 27.4 |
| Postmenopausal | 697 | 19.3 ± 8.4 | 9.7 | 13.5 | 17.7 | 23.9 | 30.4 |
Calculated LDL cholesterol calculated by Friedewald formula, % of LDLC as sdLDLC calculated using direct measurements.
Data comparing male CHD cases and controls are presented in Table 4. Men with CHD had significantly (p<0.0001) higher age and diastolic blood pressure, and were more likely to have hypertension, be on therapy for hypertension, be on aspirin, to have diabetes, to be on oral glycemic control medication or insulin, to use beta blockers, and to be on cholesterol-lowering medications than controls. With regard to percentage of men taking cholesterol-lowering medications, this value was 11.4% in control men and 46.8% in men who had heart disease. In all subjects on cholesterol lowering therapy 88% were on statin monotherapy, 5% were on statins plus another agent (mainly a fibrate), with the remainder (7%) being on either a fibrate, niacin, or resin monotherapy. Very similar distributions were seen in male and female CHD cases, as well as in male and female controls on cholesterol lowering treatment. Therefore overall 93% were receiving some form of statin therapy.
Table 4.
Characteristics and plasma lipid levels in male subjects with and without coronary heart disease participating in the Framingham Offspring Study (cycle 6)
| Variable | Coronary Heart Disease | p value for differences | |
|---|---|---|---|
| NO (n = 1335) |
YES (n = 173) |
||
|
|
|||
| Age (years) | 58.0 ± 9.7 | 65.3 ± 7.9 | <0.0001 |
| Body Mass Index (kg/m2) | 28.5 ± 4.4 | 28.7 ± 4.3 | 0.6249 |
| Body Mass Index > 30 (%) | 30.1 | 31.8 | * 0.6542 |
| Waist (cm) | 101.3 ± 10.9 | 102.4 ± 10.9 | 0.1922 |
| Waist > 102cm (%) | 42.6 | 46.2 | * 0.3669 |
| Systolic Blood Pressure (mmHg) | 129.6 ± 17.1 | 129.4 ± 17.6 | 0.8633 |
| Diastolic Blood Pressure (mmHg) | 77.7 ± 9.3 | 73.4 ± 9.5 | <0.0001 |
| Hypertension (%) | 41.9 | 66.5 | * <0.0001 |
| Hypertensive Treatment (%) | 27.7 | 58.5 | * <0.0001 |
| Taking Aspirin Regularly (%) | 31.1 | 78.0 | * <0.0001 |
| Diabetes Mellitus (%) | 11.8 | 29.5 | * <0.0001 |
| Oral Glycemic Control Drug Users (%) | 4.4 | 12.2 | * <0.0001 |
| On Insulin Treatment (%) | 1.4 | 4.6 | * 0.0029 |
| β -blocker Users (%) | 9.4 | 54.9 | * <0.0001 |
| Cigarette Smokers (%) | 14.5 | 12.7 | * 0.5190 |
| > One drink alcohol/week (%) | 54.5 | 46.8 | * 0.0563 |
| Cholesterol Lowering Drug Users (%) | 11.4 | 46.8 | * <0.0001 |
| TC (mmol/l) | 5.17 ± 0.93 | 4.81 ± 0.95 | <0.0001 |
| TG (mmol/l) | 1.33 [0.94, 1.87] | 1.43 [1.05, 2.11] | # 0.0127 |
| HDL-cholesterol (mmol/l) | 1.16 ± 0.32 | 1.04 ± 0.28 | <0.0001 |
| TC/HDLC ratio | 4.66 [3.78, 5.64] | 4.72 [3.85, 5.47] | # 0.5369 |
| non-HDL-cholesterol (mmol/l) | 4.02 ± 0.93 | 3.75 ± 0.85 | 0.0002 |
| calculated LDL-cholesterol (mmol/l) | 3.32 ± 0.82 | 2.99 ± 0.78 | <0.0001 |
| direct LDL-cholesterol (mmol/l) | 3.51 ± 0.84 | 3.22 ± 0.81 | <0.0001 |
| small dense LDL-cholesterol (mmol/l) | 0.84 ± 0.41 | 0.83 ± 0.39 | # 0.6094 |
| % of LDLC as sdLDLC | 23.7 ± 9.5 | 26.1 ± 10.0 | # 0.0019 |
| large LDL-cholesterol (mmol/l) | 2.67 ± 0.70 | 2.39 ± 0.69 | <0.0001 |
| Fasting Glucose (mmol/l) | 5.89 ± 1.49 | 6.45 ± 1.89 | 0.0003 |
| LDL-cholesterol < 2.6mmol/l (%) | 13.4 | 22.0 | * 0.0026 |
| LDL-cholesterol > 4.2mmol/l (%) | 22.4 | 10.4 | * 0.00033 |
| sdLDL-C < 0.5mmol/l (%) | 23.3 | 15.0 | * 0.0141 |
| sdLDL-C > 1.0mmol/l (%) | 27.6 | 22.0 | * 0.1157 |
| Triglyceride > 1.7mmol/l (%) | 31.1 | 38.2 | * 0.0607 |
| nonHDL-C < 3.4mmol/l (%) | 23.0 | 31.4 | * 0.0151 |
| nonHDL-C > 4.9mmol/l (%) | 15.6 | 5.3 | * 0.0003 |
Values are expressed as mean ± SD , median [25th percentile, 75th percentile], or percentage.
p-value obtained by Chi-Square test
p-value obtained by using log-transformed data
The recommended goal for patients with established CHD, based on the National Cholesterol Education Program, is an LDL-C less than 100 mg/dl or 2.6 mmol/L (25). The mean direct LDL-C value in male CHD cases was 3.2 mmol/L and only 22% of male CHD cases had an LDL-C level below the recommended target. Direct LDL-C values were used for this calculation; however very similar percentages were obtained when calculated LDL-C was used (e.g. 25%). Male CHD cases had significantly lower total cholesterol values, but their triglyceride values were higher and their HDL cholesterol values were significantly lower, as was their calculated LDL-C and their direct LDL-C than controls. Despite this, their sdLDL-C was the same as in control men, and male CHD cases had a higher percentage of LDL-C as sdLDL-C and significantly higher levels of fasting glucose than controls.
A very similar pattern was observed for the women, but the differences between cases and controls were even greater than for the men (see table 5). Female CHD cases were significantly older, had a greater mean body mass index, had a greater waist circumference, had a higher systolic blood pressure, were more likely to have hypertension, to be on antihypertensive treatment, to be taking aspirin regularly, to be diabetic, to be taking medications for diabetes, to be on beta blockers, to be on cholesterol-lowering medication, and to be postmenopausal than controls.
Table 5.
Characteristics and plasma lipid levels in female subjects with and without coronary heart disease participating in the Framingham Offspring Study (cycle 6)
| Variable | Coronary Heart Disease | p value for differences | |
|---|---|---|---|
| NO (n = 1606) |
YES (n = 74) |
||
|
|
|||
| Age (years) | 58.1 ± 9.6 | 66.1 ± 8.0 | <0.0001 |
| Body Mass Index (kg/m2) | 27.3 ± 5.7 | 29.1 ± 5.4 | 0.0082 |
| Body Mass Index > 30 (%) | 25.8 | 35.1 | * 0.0757 |
| Waist (cm) | 93.9 ± 14.8 | 101.1 ± 13.3 | <0.0001 |
| Waist > 102cm (%) | 26.3 | 39.7 | * 0.0110 |
| Systolic Blood Pressure (mmHg) | 126.5 ± 19.6 | 140.4 ± 24.6 | <0.0001 |
| Diastolic Blood Pressure (mmHg) | 73.9 ± 9.1 | 73.6 ± 11.6 | 0.8502 |
| Hypertension (%) | 35.8 | 83.8 | * <0.0001 |
| Hypertensive Treatment (%) | 22.9 | 73.0 | * <0.0001 |
| Taking Aspirin Regularly (%) | 19.8 | 62.2 | * <0.0001 |
| Diabetes Mellitus (%) | 8.3 | 29.7 | * <0.0001 |
| Oral Glycemic Control Drug Users (%) | 2.5 | 10.8 | * <0.0001 |
| On Insulin Treatment (%) | 1.2 | 9.5 | * <0.0001 |
| β -blocker Users (%) | 8.8 | 47.3 | * <0.0001 |
| On Estrogen Therapy (%) | 26.3 | 23.0 | * 0.5307 |
| Postmenopause (%) | 76.5 | 94.6 | * 0.0003 |
| Cigarette Smokers (%) | 15.5 | 18.9 | * 0.4322 |
| > One drink alcohol/week (%) | 30.5 | 21.6 | * 0.1035 |
| Cholesterol Lowering Drug Users (%) | 8.8 | 35.1 | * <0.0001 |
| TC (mmol/l) | 5.48 ± 0.99 | 5.58 ± 1.02 | 0.3887 |
| TG (mmol/l) | 1.25 [0.88, 1.84] | 1.53 [1.07, 2.05] | # 0.0030 |
| HDL-cholesterol (mmol/l) | 1.48 ± 0.41 | 1.40 ± 0.42 | 0.0725 |
| TC/HDLC ratio | 3.71 [2.99, 4.64] | 4.09 [3.47, 4.70] | # 0.0233 |
| non-HDL-cholesterol (mmol/l) | 3.98 ± 1.03 | 4.18 ± 0.99 | 0.1177 |
| calculated LDL-cholesterol (mmol/l) | 3.31 ± 0.89 | 3.33 ± 0.82 | 0.9241 |
| direct LDL-cholesterol (mmol/l) | 3.46 ± 0.93 | 3.53 ± 0.87 | 0.5430 |
| small dense LDL-cholesterol (mmol/l) | 0.68 ± 0.42 | 0.83 ± 0.44 | # 0.0015 |
| % of LDLC as sdLDLC | 19.0 ± 8.9 | 23.6 ± 12.8 | # 0.0003 |
| large LDL-cholesterol (mmol/l) | 2.79 ± 0.73 | 2.70 ± 0.78 | 0.3420 |
| Fasting Glucose (mmol/l) | 5.54 ± 1.42 | 6.54 ± 2.48 | 0.0009 |
| LDL-cholesterol < 2.6mmol/l (%) | 17.3 | 13.5 | * 0.4040 |
| LDL-cholesterol > 4.2mmol/l (%) | 21.8 | 24.3 | * 0.6068 |
| sdLDL-C < 0.5mmol/l (%) | 42.3 | 27.0 | * 0.0090 |
| sdLDL-C > 1.0mmol/l (%) | 15.3 | 27.8 | * 0.0045 |
| Triglyceride > 1.7mmol/l (%) | 29.5 | 40.5 | * 0.0417 |
| nonHDL-C < 3.4mmol/l (%) | 27.3 | 13.5 | * 0.0089 |
| nonHDL-C > 4.9mmol/l (%) | 17.5 | 13.7 | * 0.3999 |
Values are expressed as mean ± SD, median [25th percentile, 75th percentile], or percentage
p-value obtained by Chi-Square test
p-value obtained by using log-transformed data.
With regard to plasma lipid levels, despite the fact that about 4 times as many women were receiving cholesterol-lowering medication in cases than in controls (35.1% versus 8.8%), the total cholesterol levels were similar between cases and controls, and the triglyceride levels were significantly higher, as was the total cholesterol/HDL-C ratio. Calculated LDL-C and direct LDL-C levels were similar, while sdLDL-C values were significantly higher in female CHD cases than in controls. Substantially higher numbers of women with CHD had sdLDL-C over 1.0 mmol/L as compared to controls. Only 13.5% of female CHD cases were at the recommended LDL-C goal of less than 2.6 mmol/L.
Because of significant differences in use of cholesterol lowering medications between heart disease cases, we sought to determine whether such differences persisted when we excluded people who were on cholesterol-lowering medications, whether controls or heart disease patients. For men, only 92 CHD cases and 1181 controls remained, and for women 48 cases and 1464 controls remained. In these analyses no significant differences between cases and controls were observed for either men or women with regard to calculated or direct LDL-C or sdLDL-C values. It should be noted however that physicians are less likely to put CHD patients on cholesterol lowering medications if they are at or close to their LDL cholesterol goal, and the sample size was small. Prospective studies to be carried in this population in the future will provide better insight with regard to the utility of this assay for CHD risk assessment.
Discussion
Case control studies have documented that CHD cases are more likely to have elevated plasma triglyceride and sdLDL levels, and decreased HDL and large HDL levels than controls (3-9,12,13,17). The sdLDL-C assay studied here has been applied to Japanese CHD cases and controls, and increased sdLDL-C has been linked to the presence of CHD, and the severity of coronary disease as assessed by angiography (12,13). For LDL particles as assessed by gel electrophoresis, the presence of increased small dense LDL has been associated with increased CHD risk in the prospective Quebec Cardiovascular Study (21). In addition increased total LDL particle number as determined by nuclear magnetic resonance was associated with increased carotid intimal medial wall thickness in the Multi-Ethnic Study of Atherosclerosis (MESA) (22). Moreover elevated small dense LDL and total LDL particle number predicted recurrent CHD events in the Veteran Affairs HDL Intervention, and these parameters were favorably affected by gemfibrozil treatment (23). Most recently using novel ion mobility assessment of lipoprotein subspecies, three different lipoprotein patterns have been linked to CHD risk in a Swedish population: 1) increased LDL, 2) decreased HDL, and 3) increased triglycerides and small dense LDL, and decreased large HDL (24).
A link has been established between elevated triglycerides, decreased HDL, and elevated total and small dense LDL in many studies, especially in subjects with metabolic syndrome (8). Such subjects also frequently have increased waist circumference, insulin resistance, and elevated levels of C reactive protein. Giving subjects high fructose diets increases insulin resistance, visceral adiposity, CRP, triglycerides, and sdLDL-C (25). Dietary trans fatty acids also increase sdLDL-C significantly (26). In placebo controlled primary and secondary prevention statin trials, there has been great benefit in CHD risk reduction associated with LDL-C lowering, but there is still very substantial residual CHD risk (1,2). Recently in a large randomized placebo controlled trial in subjects with normal LDL –C and elevated C reactive protein levels, those receiving rosuvastatin at a dose of 20 mg/day who got their LDL-C values to below 70 mg/dl and their CRP levels to less than 1.0 mg/L, got the greatest reduction in CHD risk versus placebo (27). Rosuvastatin has also been shown to promote regression of coronary atherosclerosis (28).
There has been a substantial effort to sub-fractionate lipoprotein particles to identify those subjects with enhanced residual risk despite being on statin therapy. It remains to be determined whether these assays will be superior to the standard lipid profile in large scale prospective studies. We have previously shown that HDL particles, as assessed by two-dimensional gel electrophoresis, provide superior CHD risk prediction assessment as compared to HDL cholesterol in a case-control fashion, as well as in the prospective Veteran Affairs HDL Intervention Trial (29,30). Increasing large HDL with the simvastatin/niacin combination has been shown be predictive of regression of coronary atherosclerosis (31). The same maybe the case for sdLDL-C versus LDL-C, however this awaits prospective data analysis. We have reported that rosuvastatin at a dose of 40 mg/day, is even more effective than atorvastatin at 80 mg/day in lowering sdLDL-C by more than 50%, along with lowering LDL-C by a similar percentage (32). Rosuvastatin is also more effective in raising HDL cholesterol and large HDL particles than is atorvastatin (33).
Our data suggest that sdLDL-C offers promise as a new test for heart disease risk assessment. The advantage of this test is that it has excellent reproducibility and can be run on high throughput analyzers, which makes it much more user-friendly and more applicable than specialized tests including gradient gel electrophoresis, nuclear magnetic resonance, and gradient ultracentrifugation, which require shipping samples to specialized laboratories. Moreover this assay has now been reconfigured so that sample pre-treatment is not required prior to analysis. The gender differences are interesting, as are the differences in sdLDL-C in premenopausal versus post-menopausal women. We have previously reported similar finding for LDL-C and apoB values in this population (34). With aging and menopause there is a significant increase in LDL related to delayed clearance (35,36). Post-menopausal women also have increased apoB production into very low density lipoproteins which are converted to LDL versus premenopausal women (36). It is well known that CHD risk markedly increases with aging in both men, and after menopause in women, and alterations in LDL clearly contribute to this increased risk (20).
Our data also indicate that, despite four fold higher cholesterol lowering medication use (mainly statins) in cases than controls, mean small dense LDL cholesterol values were very similar in male cases versus controls, and were significantly higher in female cases than controls. In prospective data from Framingham we have documented that apoB is superior to calculated LDL-C and non-HDL-C in CHD risk prediction, but that the apoB/apoA-I ratio does not provide information about CHD risk than is superior to the total cholesterol/ HDL cholesterol ratio (37). It remains imperative to carry out prospective analysis to determine whether sdLDLC is an independent predictor of CHD, and how it compares with apoB levels. Ultimately the question remains whether clinicians should measure lipoprotein subclasses and apolipoproteins in their patients to optimize CHD risk. In our view emerging data indicates that these parameters do add information about residual risk, especially in patients with established CHD, but future analyses in large prospective cohort studies and intervention studies need to be carried out.
Acknowledgments
Grant/funding Support: M. Ai and S. Otokozawa were supported by research fellowships from Kyowa Medex Co, Tokyo Japan and Denka Seiken Co, Tokyo Japan, respectively. B.F. Asztalos and E.J. Schaefer were supported by grants R01 HL-60935, HL 74753 and PO50HL083813 from the National Institutes of Health and contract 53-3K – 06 from the United States Department of Agriculture Research Service. L.A. Cupples, and C. C. White were supported by NHLBI N01-HC 25195 and HL 60935 from the National Institute of Health, Bethesda, MD. Dr. Nakajima served as consultants to the Denka Seiken Corporation, Tokyo Japan, which also provided the direct LDL and small dense LDL cholesterol assay kits used in this study.
Abbreviations
- CHD
Coronary Heart Disease
- FOS
Framingham Offspring Study
- TC
Total Cholesterol
- TG
Triglyceride
- HDL-C
High Density Lipoprotein Cholesterol
- LDL-C
Low Density Lipoprotein Cholesterol
- sdLDL-C
small dense low density lipoprotein cholesterol
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