Q J Med 2002; 95: 165-171
© 2002 Association of Physicians
Abnormal low-density lipoprotein subfraction profile in patients with untreated hypertension
From the University of Birmingham Division of Medical Sciences, 1 City Hospital, and 2 Queen Elizabeth Hospital, Birmingham, UK
Received 21 May 2001 and in revised form 2 January 2002
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Background: Low-density lipoprotein (LDL) consists of a heterogeneous group of particles of varying size and electrophoretic mobility. A predominance of small, more mobile particles is a risk factor for cardiovascular disease.
Aim: To investigate the hypothesis that untreated patients with essential hypertension in the absence of vascular disease may exhibit abnormalities of LDL subfractions.
Setting: Specialist hypertension clinic.
Design: Cross-sectional study.
Methods: Following disc polyacrylamide gel electrophoresis, the mean (LDL locus) and heterogeneity (LDL spread) of mobility was recorded in 41 patients (mean age 52.6 years, 24 men) presenting with untreated essential hypertension (in the absence of vascular disease or diabetes mellitus) and in 45 healthy controls (age 56.9 years, 22 men) recruited from primary-care lists.
Results: Although there were no significant differences in total, low- or high-density lipoprotein cholesterol concentrations, LDL locus was significantly greater in the hypertensive group: mean (95%CI) 36.7 (35.7–37.6) vs. 34.8 (34.1–35.5), p=0.002. LDL locus was significantly elevated even in hypertensives with triglyceride concentrations below the median (<1.25 mmol/l). LDL spread was also greater in the hypertensive group, but not significantly: 5.6 (5.2–6.0) vs. 5.5 (5.3–5.8), p=0.10.
Discussion: Hypertensive patients have a preponderance of smaller LDL subfractions. This dyslipidaemia is not readily detected by conventional lipid assays.
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Introduction |
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Elevated low-density lipoprotein (LDL)-cholesterol concentration has been firmly established as a modifiable risk factor for cardiovascular disease.1 However, LDL consists of a heterogeneous group of particles of varying size and density.2 The smaller LDL particles may be more atherogenic for a number of reasons: they have reduced affinity for LDL receptors,3 have increased binding for endothelial proteoglycans3,4 and are better able to penetrate the arterial intima.5 In addition, these small LDL subfractions have reduced antioxidant defences and are therefore more readily oxidized by free radicals,3 leading to modification of the apolipoprotein B of LDL and rapid uptake by macrophage scavenger receptors.
Small, dense LDL subfractions have been associated with both coronary and carotid artery disease6–8 and are a risk factor for acute myocardial infarction9,10 and ischaemic heart disease events.11 For example, in the Quebec Cardiovascular Study, a prospective case-control study, the presence of small LDL particles was associated with a 3.6-fold increase in the risk of ischaemic heart disease (angina, non-fatal myocardial infarction and coronary death), independent of the potential confounding effects of diabetes, medication use and systolic blood pressure.11
Small LDL subfractions are thought to be formed by neutral lipid exchange, catalysed by cholesteryl ester transfer protein (CETP). During this process, triglycerides form chylomicron remnants, very-low- and intermediate-density lipoproteins (VLDL and IDL) are transferred to LDL in exchange for cholesteryl esters. LDL then undergoes delipidation by the enzyme hepatic lipase, resulting in smaller LDL particles.12 The principal determinant of the size of these subfractions is the serum triglyceride concentration; the higher the triglyceride concentration, the greater the proportion of smaller LDL particles. A reduction in the activity of the lipoprotein lipase enzyme on triglyceride-rich chylomicrons and VLDL results in high serum triglycerides, low HDL-cholesterol concentration and a preponderance of small LDL subfractions. This atherogenic lipid profile is characteristic of insulin-resistance and may also be associated with hypertension, although LDL subfraction profile has not previously been investigated in hypertensive patients.
We therefore hypothesized that untreated patients with essential hypertension in the absence of vascular disease may exhibit abnormalities in their LDL subfractions. To investigate this further, we studied LDL subfraction profile and serum triglyceride concentrations in untreated hypertensive patients referred to a specialist hypertension clinic who were free from overt vascular disease or diabetes mellitus.
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Methods |
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Consecutive patients with untreated essential hypertension were recruited from new referrals to a city centre teaching hospital hypertension out-patient clinic. Hypertension was defined as systolic blood pressure >160 mmHg and/or diastolic blood pressure >90 mmHg, measured by the patient's primary care physician and confirmed at the hypertension clinic on at least two separate occasions. Patients with diabetes mellitus, overt vascular disease (angina, previous myocardial infarction, coronary revascularization procedures, transient ischaemic attack, stroke, intermittent claudication, peripheral revascularization procedure or amputation for vascular disease) or renal/hepatic impairment were excluded, as were those on lipid-lowering drugs or hormone replacement therapy. Informed, written consent was obtained and the study was approved by the West Birmingham Local Research Ethics Committee.
Blood pressure was measured using a standard mercury sphygmomanometer according to the recommendations of the British Hypertension Society.13 The mean of two measurements taken 5 min apart was recorded. Blood was taken for lipid profile, including LDL subfraction analysis.
Healthy controls free from overt vascular disease (as above) were recruited from two local primary-care centres, as part of a ‘health screen’ programme. These patients were ‘healthy’ by careful history and examination, and thus, those with a history of hypertension, vascular disease or diabetes mellitus, those receiving lipid-lowering drugs and those with blood pressure >160/90 mmHg were excluded.
Biochemical methods
Blood was taken into Vacutainers containing clotting activator (Beckton-Dickenson) and allowed to clot. Following separation by centrifugation (3000 g for 15 min at 4 °C), serum was stored at -80 °C. LDL subfraction profile was then analysed by disc polyacrylamide gel electrophoresis (DPAGE) using the Lipoprint system (Quantimetrix). Thawed serum (25 µl) was added to each gel tube (pre-cast polyacrylamide, 3%). To this 200 µl loading gel (2.4 g/dl acrylamide, 0.2 g/dl N,N methlyenebisacrylamide, 3.6 mg/dl Sudan Black B) was added. Following repeated inversion of the tubes to allow thorough mixing, the gels were allowed to photopolymerize for 30 min in front of a fluorescent light source. Electrophoresis was then done at a constant current of 3 mA per tube for 60 to 70 min, until the HDL fraction had migrated approximately 40 mm. The electrophoresis buffer contained 66.1% tris (hydroxymethyl)aminomethane and 33.9% boric acid (pH 8.2–8.6). Gel tubes were allowed to stand for at least 30 min and then scanned directly at a wavelength of 610 nm (ImageMater Software, Pharmacia). The resulting digitized images were exported to a bespoke computer program. This program uses the observation that the LDL concentration profile approximates to a normal distribution curve and can thus be descrribed in terms of the mean (locus) and standard deviation (spread) of LDL mobility. The peaks of VLDL and HDL serve as reference points to which the mobility of LDL can be compared (Figure 1
). DPAGE of lipoproteins was originally described by Naito in 197314 and has been used to describe abnormalities of LDL subfraction profile in a number of disease states.6,8,15 The use of dedicated computer software allows more precise reporting of the electrophoresis results. Within-batch coefficients of variation were 2.4% for LDL locus and 8.2% for LDL spread. Between-batch coefficients of variation were 3.3% for LDL locus and 8.1% for LDL spread.
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Total serum cholesterol and serum triglyceride concentrations were measured by standard enzymic techniques in the Department of Biochemistry, City Hospital, Birmingham. HDL-cholesterol was measured by dextran sulphate–magnesium chloride precipitation in the Clinical Investigation Unit, University of Birmingham. LDL-cholesterol concentration was calculated using the Friedewald formula for those with triglycerides <4.5 mmol/l.16
Statistical methods
Differences in demographics were assessed using 
2 and unpaired t-tests as appropriate. Body mass index, HDL-cholesterol, serum triglyceride concentration, LDL locus and LDL spread were found to be positively skewed and were therefore log-transformed before statistical comparison. Results were then expressed in natural units for ease of interpretation. Backward logistic regression analysis was used to identify factors independently associated with the presence of hypertension. Pearson's correlation coefficients and linear regression were used to investigate the association of blood pressure, standard lipid parameters with LDL locus and LDL spread.
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Results |
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We recruited 41 patients with untreated essential hypertension and 45 healthy controls (Table 1
). Body mass index (Table 1 ) and serum triglyceride concentration were significantly greater in the hypertensive group, whilst total, HDL- and LDL-cholesterol concentrations were similar in the two groups (Table 2). LDL locus was significantly greater in the hypertensive group. LDL spread showed a non-significant trend towards being greater in the hypertensives.
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Gender, age, body mass index, glucose, triglycerides, LDL- and HDL-cholesterol concentrations, LDL locus and LDL spread were entered into a stepwise logistic regression model (Table 3
, Model 1). Body mass index and LDL locus were the only significant independent associates of hypertension. An increase in LDL locus of 1 SD was associated with an adjusted OR of 2.18 for hypertension. In a second logistic regression model in which LDL spread but not LDL locus was entered, body mass index and plasma glucose concentration (borderline significance) were the only independent associates of hypertension (Table 3, Model 2).
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Figure 2
shows the difference in LDL locus in patients with normal (below the median, <1.25 mmol/l) and moderately raised (above the median, >1.25 mmol/l) triglyceride concentrations. In those with moderately raised triglyceride concentrations, LDL locus was similar in hypertensives and controls. However, in those with lower triglyceride concentrations, LDL locus was significantly greater in hypertensives. LDL spread was similar in hypertensives and controls regardless of serum triglyceride concentration.
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In the hypertensives, both LDL locus and LDL spread correlated with elevated serum triglyceride concentration (Figures 3
and 4). There were no significant correlations with HDL-cholesterol concentration in either group. Log LDL locus correlated with systolic blood pressure (r=0.23, p=0.04) and diastolic blood pressure (r=0.29, p=0.006), but not pulse pressure (r=-0.09, p=NS); whilst log LDL spread did not correlate with systolic (r=0.10), diastolic (r=0.04) or pulse (r=-0.07) pressures (all p=NS). Multivariate linear regression models that included age, sex, log body mass index, log triglyceride concentration and log HDL-cholesterol concentration explained less than half the variance in LDL locus or LDL spread.
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Discussion |
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A number of previous studies in patients with hypertension have observed an aggregation of metabolic abnormalities, including hypertriglyceridaemia, low HDL-cholesterol concentration, insulin resistance and central obesity.17 Other studies have shown an increased proportion of small LDL subfractions in patients with familial dyslipidaemic hypertension.18,19 However, this study demonstrates abnormalities of LDL subfraction profile in patients with uncomplicated essential hypertension who have normal levels of serum triglycerides and HDL-cholesterol. The observed difference in LDL locus indicates that LDL particles are on average smaller in hypertensive patients, and there is a suggestion that they may also be more heterogeneous (increased LDL spread). Adjustment for gender, age, glucose, body mass index and conventional lipid parameters does not alter this finding. Whilst both patients and controls were identified as having no overt vascular disease, it is possible that subclinical (‘silent’) atherosclerosis may have been present in a number of patients in either group.
As expected, in the control subjects there was a significant correlation between abnormal LDL profile and higher (but still modest) concentration of serum triglycerides. Surprisingly, this was not observed in the patients with hypertension in whom the LDL profile was abnormal, even in those with low triglyceride concentration (<1.25 mmol/l). One possible explanation of this is that serum triglyceride concentration may be an inadequate measure of chylomicron, VLDL and IDL metabolism, and that reduced lipoprotein lipase activity leads to decreased clearance of chylomicron remnants, resulting in enhanced neutral lipid exchange. Alternatively, an increase in the activity of cholesteryl ester transfer protein and/or hepatic lipase may lead to a more rapid triglyceride-enrichment and subsequent delipidation of LDL.12 In this population, it does not appear to be possible to predict LDL profile on the basis of conventional lipid measurements.
The finding of abnormal LDL subfraction profiles in patients with uncomplicated hypertension has a number of potential implications for clinical management. A key component of the assessment of a patient with hypertension is the estimation of cardiovascular risk. Small LDL subfractions have been found to be a risk factor for coronary artery disease in prospective case-control studies,9–11 but it is unclear whether this is independent of conventional lipid measurements. At present, there is no well-validated risk prediction model that incorporates abnormalities of LDL subfraction profile.
It is important to consider whether the documented abnormalities of LDL profile require specific treatment. At present there is no direct evidence to support the hypothesis that modification of LDL profile will reduce cardiovascular morbidity and mortality. However, fibrates do improve the atherogenic lipid profile, lowering serum triglycerides, raising HDL-cholesterol concentration and increasing LDL particle size.20,21 Fibrates may also reduce cardiovascular morbidity and mortality, particularly in those with low levels of HDL-cholesterol and elevated serum triglyeride concentration.22 The Veterans Affairs High-Density Lipoprotein Intervention Trial (VA-HIT) showed a significant 22% relative risk reduction in coronary events in response to treatment with gemfibrozil following myocardial infarction. These men had normal to low levels of LDL-cholesterol (<3.6 mmol/l) which did not change significantly in response to treatment. By contrast, serum triglyceride concentration was reduced by 31%.23 The question remains whether treatment with a fibrate should be initiated in individuals with abnormal LDL subfraction profiles but ‘normal’ levels of triglyceride and HDL-cholesterol and no clinical evidence of vascular disease. However, this hypothesis can only be addressed by large-scale randomized controlled trials.
An alternative lipid-lowering strategy would be to use an HMG-CoA reductase inhibitor (a ‘statin’). There is considerable evidence that these drugs can reduce cardiovascular mortality in both primary and secondary prevention settings and over a wide range of baseline total cholesterol concentrations.24–28 Statins have relatively little impact on triglyceride metabolism, except in high doses or in those with substantially elevated baseline triglyceride concentrations,29 but there is some evidence that they may perhaps improve LDL subfraction profile.30 However, it is not known whether treatment with a statin would be more or less effective than treatment with a fibrate in preventing cardiovascular events in patients with ‘normal’ serum lipid concentrations but abnormal LDL subfraction profile.
This study is limited by its cross-sectional nature. Further, we cannot account for all possible confounders on the measured indices. For example, the size and nature of this study would not be able to precisely address the influence of (say) age, diet, exercise, social class, smoking, etc., or their interactions. Furthermore, the relatively small size and the fact that our study is not population-based, and based on referrals to our hypertension clinic, we cannot be certain of the generalizability to the general population.
In conclusion, this study shows that LDL subfraction profile may be deranged in patients with uncomplicated essential hypertension in the absence of vascular disease at the time of presentation. This may have significant implications for the selection of both antihypertensive and lipid-lowering treatments in these patients.
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Notes |
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Address correspondence to Professor G.Y.H. Lip, University Department of Medicine, City Hospital, Birmingham B18 7QH. e-mail: g.y.h.lip{at}bham.ac.uk
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