Low Density Lipoprotein Subspecies Research Articles

Pathophysiology of Diabetic Dyslipidemia

Accumulating clinical evidence has suggested serum triglyceride (TG) is a leading predictor of atherosclerotic cardiovascular disease, comparable to low-density lipoprotein (LDL)-cholesterol (C) in populations with type 2 diabetes, which exceeds the predictive power of hemoglobinA1c. Atherogenic dyslipidemia in diabetes consists of elevated serum concentrations of TG-rich lipoproteins (TRLs), a high prevalence of small dense low-density lipoprotein (LDL), and low concentrations of cholesterol-rich high-density lipoprotein (HDL)2-C. A central lipoprotein abnormality is an increase in large TG-rich very-low-density lipoprotein (VLDL)1, and other lipoprotein abnormalities are metabolically linked to increased TRLs. Insulin critically regulates serum VLDL concentrations by suppressing hepatic VLDL production and stimulating VLDL removal by activation of lipoprotein lipase. It is still debated whether hyperinsulinemia compensatory for insulin resistance is causally associated with the overproduction of VLDL. This review introduces experimental and clinical observations revealing that insulin resistance, but not hyperinsulinemia stimulates hepatic VLDL production. LDL and HDL consist of heterogeneous particles with different size and density. Cholesterol-depleted small dense LDL and cholesterol-rich HDL2 subspecies are particularly affected by insulin resistance and can be named “Metabolic LDL and HDL,” respectively. We established the direct assays for quantifying small dense LDL-C and small dense HDL(HDL3)-C, respectively. Subtracting HDL3-C from HDL-C gives HDL2-C. I will explain clinical relevance of measurements of LDL and HDL subspecies determined by our assays. Diabetic kidney disease (DKD) substantially worsens plasma lipid profile thereby potentiated atherogenic risk. Finally, I briefly overview pathophysiology of dyslipidemia associated with DKD, which has not been so much taken up by other review articles.

A Review of Time Courses and Predictors of Lipid Changes with Fenofibric Acid-Statin Combination

Fibrates activate peroxisome proliferator activated receptor α and exert beneficial effects on triglycerides, high-density lipoprotein cholesterol, and low density lipoprotein subspecies. Fenofibric acid (FA) has been studied in a large number of patients with mixed dyslipidemia, combined with a low- or moderate-dose statin. The combination of FA with simvastatin, atorvastatin and rosuvastatin resulted in greater improvement of the overall lipid profile compared with the corresponding statin dose. The long-term efficacy of FA combined with low- or moderate- dose statin has been demonstrated in a wide range of patients, including patients with type 2 diabetes mellitus, metabolic syndrome, or elderly subjects. The FA and statin combination seems to be a reasonable option to further reduce cardiovascular risk in high-risk populations, although trials examining cardiovascular disease events are missing.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Alterations of lipids and apolipoprotein CIII in very low density lipoprotein subspecies in type 2 diabetes

Very low density lipoprotein (VLDL) particles are heterogeneous, comprising two main subspecies, VLDL 1 (Sf 60-400) and VLDL 2 (Sf 20-60). The aim of the study was to examine the distribution and composition of VLDL subspecies in type 2 diabetes. We studied the composition and concentration of triglyceride-rich lipoproteins (TRLs) in 217 type 2 diabetic patients and 93 control subjects between 50 and 75 years of age. Lipoprotein subspecies were separated by density-gradient ultracentrifugation. Apolipoprotein (apo) CIII and apo E in plasma and apo CIII in TRL subspecies were measured by nephelometry and apo CII in serum by a commercial kit using a single radial immunodiffusion method. The concentrations of VLDL 1, VLDL 2 and intermediate density lipoprotein were significantly increased in type 2 diabetes subjects, the change being most marked for VLDL 1. There was a strong linear correlation between VLDL 1 triglycerides and plasma triglycerides in both groups (r = 0.879, p < 0.001 and r = 0.899, p < 0.001). Diabetic subjects had markedly higher plasma ratios of apo CII:apo CIII and apo CIII:apo E. Despite elevated plasma apo CIII, type 2 diabetic subjects had a relative deficiency of apo CIII in all TRL subspecies, suggesting profound disturbances of apo CIII metabolism. The elevation of VLDL 1 triglycerides is the major determinant of plasma triglyceride concentration in normal subjects and in type 2 diabetic individuals. Both apo CIII and apo E metabolism are disturbed in type 2 diabetes.

Effects on lipoprotein subclasses of combined expression of human hepatic lipase and human apoB in transgenic rabbits.

The effects of combined expression of human hepatic lipase (HL) and human apolipoprotein B (apoB) on low-density lipoprotein (LDL) subclasses were examined in rabbits, a species naturally deficient in HL activity. In apoB-transgenic rabbit plasma, >80% of the protein was found in the 1.006- to 1.050-g/mL fraction. Gradient gel electrophoresis (GGE) of this fraction revealed two distinct species, designated large and small LDL. A denser fraction (d=1.050 to 1.063 g/mL) contained small LDL as well as another discrete LDL subspecies, designated very small LDL. Expression of HL resulted in reductions in protein concentrations in the 1.006- to 1.050-g/mL density-gradient subfractions containing large (6.5+/-4.1 versus 32.6+/-12.0 mg/dL, P<0.005) and small LDL (59.6+/-17.4 versus 204.3+/-50.3 mg/dL, P<0.002). A concomitant small but not significant increase in protein concentration in the denser LDL fraction (48.0+/-28.2 versus 44.6+/-18.2 mg/dL) was due primarily to an increase in very small LDL (25.9+/-3.1 versus 9.6+/-5.4% of total LDL GGE densitometric area, P<0.002). These findings support a direct role for HL in regulating total plasma LDL concentrations as well as in the production of smaller, denser LDL from larger, more buoyant precursors.

Comparison of effectiveness of statin monotherapy versus statin and niacin combination therapy in primary prevention and effects on calcified plaque burden

T combination of a statin and niacin is increasingly being prescribed for treatment of combined lipid disorders, and has recently been shown to produce a dramatic decrease in clinical events and atherosclerosis progression, measured by quantitative coronary angiography in secondary prevention patients.1 This study was designed to determine whether calcified plaque progression, determined by electron beam tomography (EBT), differs in groups of primary prevention patients treated with statins alone compared with statin and niacin combination therapy. In addition, the correlations between the change in calcified plaque burden and changes in low-density lipoprotein (LDL) particle size and subgroups were evaluated. • • • One hundred sixty-two consecutive asymptomatic patients with EBT evidence for subclinical atherosclerosis, who were treated with a statin alone or in combination with niacin (Niaspan; Kos Pharmaceuticals, Miami, Florida) and underwent repeat EBT evaluation, comprised the patient population. This was an observational study; serial EBT evaluation and lipidlowering agents and doses employed were chosen by the treating physicians based on clinical considerations, and not according to criteria set by the investigators. All patients gave consent for their data to be used in subsequent analyses. No patient had a history of clinical coronary artery disease. Hypertension was defined as the current or recommended use of antihypertensive medications for blood pressure control. Smoking was defined as current tobacco usage, and diabetes was defined as the current or recommended use of diet or medication to decrease blood sugar. Family history of coronary artery disease was defined as established disease in first-degree male relatives 56 years of age or female relatives 66 years of age. Body mass index was calculated as weight (kilograms)/height (meters squared). Lipid measurements were obtained at the time of both EBT evaluations. EBT was performed using an Imatron C-150 scanner (GE/Imatron, South San Francisco, California) and previously described acquisition protocol.2 The coronary artery calcium score was calculated with the Agatston method.3 The calcium volume score, a parameter of plaque burden that is density independent, was calculated according to the method of Callister et al.4 The calcium percentile, an age and gender corrected index of plaque prematurity, was derived from the University of Illinois asymptomatic patient database. EBT studies done before and after treatment were reviewed side-by-side for comparability of analysis by an experienced observer (HSH), blinded to the treatment effects. Changes in coronary calcium and calcium volume scores were calculated on an absolute basis and as a percent change per year. To avoid skewing the data by large percentage changes in a few patients with lower scores, the percent change per year for the treated and untreated groups was calculated by dividing the mean annualized change by the mean baseline values for the groups as a whole, rather than by the mean of the percent change per year for each patient. Fasting plasma lipid and lipoprotein cholesterol concentrations were determined by the methods of the Lipid Research Clinics in all patients.5 In 53 patients treated with niacin, densitometric measurements of LDL subspecies were carried out using custom made, triple gradient (S3), 2% to 16% polyacrylamide gradient gel electrophoresis (S3GGE) of plasma samples (Berkeley HeartLab, Burlingame, California).6 LDL peak particle diameter was measured and LDL subclass distribution was determined in 3 large regions (I, IIa, IIb) and 4 small regions (IIIa, IIIb, IVa, IVb). LDL subclass pattern was classified based on LDL peak particle diameter, number and position of peaks, and From the Beth Israel Medical Center, New York, New York; and Kronos Longevity Research Institute, Phoenix, Arizona. Dr. Hecht’s address is: PO Box 450, 9 Woodland Road, Brookside, New Jersey 07926. E-mail: hhecht@aol.com. Manuscript received July 22, 2002; revised manuscript received and accepted September 20, 2002. TABLE 1 Patient Characteristics

Mildly oxidized LDL particle subspecies are distinct in their capacity to induce apoptosis in endothelial cells: role of lipid hydroperoxides.

The risk of atherosclerosis is intimately related to the heterogeneity of low-density lipoprotein (LDL) particles. The potential relationship between oxidative modification of distinct LDL subspecies and induction of apoptosis in arterial wall cells is indeterminate. The capacity of light LDL3 versus dense LDL5 to induce cytotoxicity in endothelial cells as a function of the degree of copper-mediated oxidation was compared. Mildly oxidized LDL3 (oxLDL3) exerted potent cytotoxicity, which was intimately related to both the degree of oxidation and the oxLDL3 concentration based on either cholesterol content or particle number. In contrast, dense LDL5 particles exerted a minor effect on cell viability. Cells incubated with oxLDL3 exhibited apoptotic features, with cytoplasmic condensation, cell or nuclear fragmentation, and accumulation of DNA fragments. OxLDL3-induced apoptosis involved cytoplasmic release of cytochrome c, with a concomitant increase in caspase-3-like protease activity. OxLDL3 particles were uniquely distinct from oxLDL5 particles in their elevated content of lipid hydroperoxides. Hydroperoxide removal by NaBH4 markedly reduced oxLDL3-induced cytotoxicity, leading to an increase in cell viability. Lipid hydroperoxide content of oxidatively modified LDL subclasses is therefore a major determinant of the induction of apoptosis in endothelial cells. These data are highly relevant to atherogenic hypercholesterolemia, in which the LDL phenotype is dominated by elevated concentrations of light LDL3.

LDL particle size: Relation to risk factors and subclinical atherosclerosis measured by coronary calcium

The effect of ciprofibrate treatment on the atherogenic profile of low-density lipoprotein (LDL) subspecies in combined hyperlipidemia (CHL) has been investigated in six patients displaying elevated plasma triglyceride and cholesterol levels ( > 200 and > 250 mg/dl, respectively). The E2E2 phenotype was excluded; four patients possessed familial antecedents of premature coronary heart disease (CHD). Analysis of five LDL subclasses separated by isopycnic density gradient ultracentrifugation showed a predominance of dense LDL subspecies (LDL-4 and LDL-5, d 1.039–1.063 g/ml; 51% of total LDL mass) in the asymmetric LDL density profile characteristic of CHL patients at baseline. Ciprofibrate treatment (100 mg/day for 1 month) effected marked reductions in both total plasma LDL and apo B-100 levels ( ∼ 19% and ∼ 23%, respectively). Equally, the plasma profile of LDL subspecies was normalised to a significant degree as a result of preferential reduction in the elevated levels of both dense subspecies (LDL-4 and LDL-5; −43% and −54%, respectively; P < 0.03 and P < 0.06, respectively). The circulating concentrations of light LDL (LDL-1, d 1.019-1.023 g/ml) were also diminished significantly by ciprofibrate (−30%; P < 0.06). Furthermore, ciprofibrate not only effected reductions in the elevated triglyceride content of the hydrophobic core of all LDL subspecies but also normalised their common deficiency in free cholesterol. In addition, the abnormally small particle diameters of LDL-4 and −5 were increased to normal. Plasma levels of both apo B-100 and triglycerides were significantly and positively correlated with those of LDL-4 and LDL-5, suggesting not only that the degree of triglyceride elevation is intimately linked to the extent of shift in LDL subclass profile towards denser subspecies, but also that triglyceride reduction upon treatment strongly influences LDL-4 and LDL-5. In conclusion, our findings indicate that ciprofibrate treatment in combined hyperlipidemia results in marked reduction in plasma triglyceride levels (-33%), and that such reduction is intimately linked to normalisation of both the qualitative and quantitative features of the atherogenic LDL subspecies profile typical of this disorder.

Contribution of hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein to LDL and HDL heterogeneity in healthy women.

Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) have been independently associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) size in different cohorts. These studies have been conducted mainly in men and in subjects with dyslipidemia. Ours is a comprehensive study of the proposed biochemical determinants (lipoprotein lipase, HL, CETP, and triglycerides) and genetic determinants (HL gene [LIPC] and Taq1B) of small dense LDL (sdLDL) and HDL subspecies in a large cohort of 120 normolipidemic, nondiabetic, premenopausal women. HL (P<0.001) and lipoprotein lipase activities (P=0.006) were independently associated with LDL buoyancy, whereas CETP (P=0.76) and triglycerides (P=0.06) were not. The women with more sdLDL had higher HL activity (P=0.007), lower HDL2 cholesterol (P<0.001), and lower frequency of the HL (LIPC) T allele (P=0.034) than did the women with buoyant LDL. The LIPC variant was associated with HL activity (P<0.001), HDL2 cholesterol (P=0.034), and LDL buoyancy (P=0.03), whereas the Taq1B polymorphism in the CETP gene was associated with CETP mass (P=0.002) and HDL3 cholesterol (P=0.039). These results suggest that HL activity and HL gene promoter polymorphism play a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2 cholesterol in normal physiological states as well as in the pathogenesis of various disease processes.

Different susceptibility to oxidation of proline and arginine residues of apolipoprotein B-100 among subspecies of low density lipoproteins

γ-Glutamyl semialdehyde is a primary oxidation product of apolipoprotein (apo) B-100 proline (Pro) and arginine (Arg) side chain residues. By reduction γ-glutamyl semialdehyde forms 5-hydroxy-2-aminovaleric acid (HAVA). Here we describe the application of sensitive and specific HAVA measurement to characterize the formation of γ-glutamyl semialdehyde in several domains of apoB-100 in LDL 1 (S f 7–12) and LDL 2 (S f 0–7) subfractions subjected to oxidative damage in the presence of iron in vitro. Results suggest that susceptibility of apoB-100 Pro and Arg residues toward oxygen radicals drastically changes along the lipoprotein metabolic cascade.

Action of atorvastatin in combined hyperlipidemia : preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles.

Combined hyperlipidemia (CHL) is characterized by a concomitant elevation of plasma levels of triglyceride-rich, very low density lipoproteins (VLDLs) and cholesterol-rich, low density lipoproteins (LDLs). The predominance of small, dense LDLs contributes significantly to the premature development of coronary artery disease in patients with this atherogenic dyslipoproteinemia. In the present study, we evaluated the impact of atorvastatin, a newly developed inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, on the cholesteryl ester transfer protein (CETP)-mediated remodeling of apolipoprotein (apo) B-containing lipoprotein subspecies, and more specifically, the particle subpopulations of VLDL and LDL in CHL. In parallel, we evaluated the atorvastatin-induced modulation of the quantitative and qualitative features of atherogenic apo B-containing and cardioprotective apo AI-containing lipoprotein subspecies. Atorvastatin therapy (10 mg/d for a 6-week period) in patients with a lipid phenotype typical of CHL (n=18) induced reductions of 31% (P<0.0001) and 36% (P<0.0001) in plasma total cholesterol and LDL cholesterol, respectively. In addition, atorvastatin significantly reduced VLDL cholesterol, triglycerides, and apo B levels by 43% (P<0.0001), 27% (P=0.0006), and 31% (P<0.0001), respectively. The plasma concentrations of triglyceride-rich lipoproteins (VLDL1, Sf 60 to 400; VLDL2, Sf 20 to 60; and intermediate density lipoproteins, Sf 12 to 20) and of LDL, as determined by chemical analysis, were markedly diminished after drug therapy (-30% and -28%, respectively; P<0.0007). Atorvastatin significantly reduced circulating levels of all major LDL subspecies, ie, light (-28%, P<0.0008), intermediate (-27%, P<0.0008), and dense (-32%, P<0.0008) LDL; moreover, in terms of absolute lipoprotein mass, the reduction in dense LDL levels (mean -62 mg/dL) was preponderant. In addition, the reduction in plasma dense LDL concentration after therapy was significantly correlated with a reduction in plasma VLDL1 levels (r=0.429, P=0.0218). Atorvastatin induced a significant reduction (-7%, P=0.0039) in total CETP-dependent CET activity, which accurately reflects a reduction in plasma CETP mass concentration. Total CETP-mediated CET from high density lipoproteins to apo B-containing lipoproteins was significantly reduced (-26%, P<0.0001) with drug therapy. Furthermore, CETP activity was significantly correlated with the atorvastatin-induced reduction in plasma VLDL1 levels (r=0.456, P=0. 0138). Indeed, atorvastatin significantly and preferentially decreased CET from HDL to the VLDL1 subfraction (-37%, P=0.0064), thereby reducing both the levels (-37%, P=0.0001) and the CE content (-20%, P<0.005) of VLDL1. We interpret our data to indicate that 2 independent but complementary mechanisms may be operative in the atorvastatin-induced reduction of atherogenic LDL levels in CHL: first, a significant degree of normalization of both the circulating levels and the quality of their key precursors, ie, VLDL1, and second, enhanced catabolism of the major LDL particle subclasses (ie, light, intermediate, and dense LDL) due to upregulation of hepatic LDL receptors.

LDL particle size distribution is associated with carotid intima-media thickness in healthy 50-year-old men.

Results of cross-sectional and prospective studies have suggested that small, dense low-density lipoprotein (LDL) particles predispose to coronary heart disease. We investigated the relationships between plasma concentrations of LDL subfractions and intima-media thickness (IMT) of the common carotid artery (CCA), quantified by B-mode ultrasound, in 94 healthy, 50-year-old men, all of whom were homozygous for the apolipoprotein E3 allele. A novel 3% to 7.5% polyacrylamide gradient gel was developed to provide separation of LDL subfractions with high resolution, as was a procedure to quantify plasma concentrations of these LDL subspecies. The LDL particle size distribution pattern obtained by the gradient gel electrophoresis procedure was in good agreement with the one obtained by a well-established, single-spin density gradient ultracentrifugation technique. LDL-II (particle size, 23.5 to 25.0 nm) was the most abundant subfraction, and its plasma concentration correlated closely with the total LDL cholesterol concentration (r=0. 61, P<0.001) but not with CCA IMT (r=-0.13, NS). In contrast, the plasma concentration of the predominant small, dense LDL particle subfraction (LDL-III; particle size, 22.5 to 23.5 nm) correlated strongly with CCA IMT (r=0.42, P<0.001). In multivariate analysis, the plasma concentration of the LDL-III subfraction contributed significantly to the variation in CCA IMT (R(2)=0.19). When plasma triglycerides and LDL cholesterol were forced into the multivariate model, 10% of the variation in CCA IMT was still accounted for by the LDL-III subfraction. In summary, use of a novel and sensitive gradient gel electrophoresis method for evaluation of LDL heterogeneity provided the basis for demonstrating an independent relation between the plasma concentration of small LDL and IMT of the CCA in healthy, middle-aged men.

LDL particle size: an important drug target?

Low density lipoprotein (LDL) is the main carrier of plasma cholesterol and a major component of atherosclerotic plaque [1]. Lowering LDL cholesterol reduces coronary events and mortality from coronary artery disease (CAD) [2–4], however, the relation between LDL cholesterol concentration and (CAD) is complex. Many patients with CAD have plasma LDL cholesterol concentrations in the normal rangefor the general population [5]. Thus, it could be that coronary risk goes beyond LDL cholesterol concentration to the characteristics of the LDL particles themselves. The purpose of this communication is to address the issue of whether LDL particle size and density influences its atherogenecity and how this might be modified by drug therapy.

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

Microphase Separation in Low Density Lipoproteins: EVIDENCE FOR A FLUID TRIGLYCERIDE CORE BELOW THE LIPID MELTING TRANSITION

The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1. 0244-1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 degreesC, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core.

Apolipoprotein B-100 conformation and particle surface charge in human LDL subspecies: implication for LDL receptor interaction.

The plasma low-density lipoprotein (LDL) profile in coronary artery disease patients is characterized by a predominance of small, dense LDL. Small, dense LDL exhibit both high susceptibility to oxidation and low binding affinity for the LDL receptor, suggesting that these particles may be of elevated atherogenic potential. Here we examine whether the variation in biological function is due to differences in apo B-100 conformation that alter the interaction with the cellular LDL receptor. The microenvironments (pKa) of Lys residues in apo B-100 in small, dense, intermediate, and light human LDL subspecies have been compared by 13C NMR, and the net surface charge of these particles has been characterized. Relative to the total LDL fraction, small, dense, and light LDL subspecies have a decreased number of pKa 8.9 Lys, while intermediate density LDL has a consistently higher number of pKa 8.9 Lys. It follows that differences in protein conformation, as reflected in the Lys microenvironments, exist in the different LDL subspecies. Electrophoretic mobility measurements revealed that the light LDL subfractions exhibit a surface charge at pH 8.6 that is from -26 to -34e more negative than the intermediate density LDL subfraction. For the small, dense LDL particles the increments in negative charge range from -7 to -17e relative to the intermediate density LDL subfraction. These results suggest that differences in the conformation of apo B-100 and surface charge between LDL subspecies are major determinants of their catabolic fate. The lower number of pKa 8.9 Lys leads to a reduction in binding of small, dense, and light LDL to the cellular LDL receptor and prolongs their plasma residence time, thereby elevating the atherogenicity of these particles. These data support the proposal that the intermediate LDL subspecies constitute the optimal ligand for the LDL receptor among human LDL particle subpopulations.

Differences in LDL subspecies involve alterations in lipid composition and conformational changes in apolipoprotein B

In order to investigate causes of variability in low density lipoprotein (LDL) particle size, we have assessed LDL composition in plasma from 66 subjects, each with a single LDL band, by 2-16% gradient gel electrophoresis, with a total of eight discrete sizes (LDL-1 to LDL-8). Lipoprotein concentrations were analyzed by standard methods; specific proteins were assessed by immunoassay and electrophoresis. Results showed decreased anhydrous molecular weight with size (2.67 +/- 0.07 x 10(6) to 1.78 +/- 0.19 x 10(6)), along with decreased relative content for cholesteryl ester (41.5% to 24.3%), free cholesterol (10.1% to 4.6%), and phospholipid (23.7% to 18.9%), and increased triglyceride (4.1% to 21.0%) and protein (20.5% to 31.2%) content. As LDL size decreased, the ratio of surface cholesterol to phospholipid decreased from 0.53 to 0.29, and the fraction of surface area covered by lipid decreased from 0.74 to 0.47. Moreover, core volume decreased with size from 24.2 A3 x 10(5) to 15.9 A3 x 10(5), and the ratio of surface-to-core lipids fell from 0.59 to 0.46. Based on surface pressures of 30 mN/m, the area covered by surface lipid was calculated to range from 6.45 A2 x 10(4) in the largest LDL, to 3.10 A2 x 10(4) in the smallest. Computer modeling indicates that alterations in the tertiary structure of apoB-100 are required to account for surface changes. The estimated core surface area requiring coverage by apoB increased with decreasing particle size from 2.26 A2 x 10(4) to 3.46 A2 x 10(4). To accommodate coverage of increasing relative surface area associated with decreasing size, apoB thickness at the interface was calculated to decrease from approximately 25 A to 16 A. Such conformational changes in apoB may alter exposed epitopes, possibly causing changes in LDL receptor binding affinity and resistance to oxidation.

The atherogenic role of triglycerides and small, dense low density lipoproteins: impact of ciprofibrate therapy

Triglyceride levels greater than 150 mg/dl are associated with the atherogenic lipoprotein phenotype, represented by a predominance of small, dense low density lipoproteins (LDL) and diminished concentrations of high density lipoproteins. This phenotype is characteristic of patients with combined hyperlipidemia (CHL). We evaluated the impact of ciprofibrate therapy (100 mg daily) for 1 month on the quantitative and qualitative characteristics of the LDL particle profile in CHL patients ( n = 9). Marked reductions in plasma levels of triglycerides (−33%), cholesterol (−15.5%), LDL-cholesterol (−15.2%) and apolipoprotein-B (−22.7%) were accompanied by a significant degree of normalisation in the LDL subspecies profile; such normalisation resulted from a preferential reduction in the elevated levels of dense LDL subspecies (LDL-4 and LDL-5, −43% and −54%, respectively; P < 0.03 and P < 0.006, respectively). Concentrations of light LDL (LDL-1) were also reduced significantly (−30%, P < 0.006), while those of LDL-3 of intermediate density ( d = 1.029–1.039 g/ml) were moderately increased (+23%). The ciprofibate-induced normalisation of both the quantitative and qualitative features of the atherogenic LDL particle profile characteristic of combined hyperlipidemia is consistent with a reduction in the elevated cardiovascular risk in this patient group.

The atherogenic role of triglycerides and small, dense low density lipoproteins: impact of ciprofibrate therapy

Triglyceride levels greater than 150 mg/dl are associated with the atherogenic lipoprotein phenotype, represented by a predominance of small, dense low density lipoproteins (LDL) and diminished concentrations of high density lipoproteins. This phenotype is characteristic of patients with combined hyperlipidemia (CHL). We evaluated the impact of ciprofibrate therapy (100 mg daily) for 1 month on the quantitative and qualitative characteristics of the LDL particle profile in CHL patients (n = 9). Marked reductions in plasma levels of triglycerides (−33%), cholesterol (−15.5%), LDL-cholesterol (−15.2%) and apolipoprotein-B (−22.7%) were accompanied by a significant degree of normalisation in the LDL subspecies profile; such normalisation resulted from a preferential reduction in the elevated levels of dense LDL subspecies (LDL-4 and LDL-5, −43% and −54%, respectively; P < 0.03 and P < 0.006, respectively). Concentrations of light LDL (LDL-1) were also reduced significantly (−30%, P < 0.006), while those of LDL-3 of intermediate density (d = 1.029–1.039 g/ml) were moderately increased (+23%). The ciprofibate-induced normalisation of both the quantitative and qualitative features of the atherogenic LDL particle profile characteristic of combined hyperlipidemia is consistent with a reduction in the elevated cardiovascular risk in this patient group.

Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects

Plasma cholesterol level is controlled by various factors. In the present study, high plasma activity of cholesterol esterase was found to correlate with plasma total cholesterol and low density lipoprotein (LDL) cholesterol levels in normolipidemic human subjects. However, the cholesterol esterase is not elevated in plasma of patients with familial hypercholesterolemia. These observations suggest that cholesterol esterase level is not determined by plasma cholesterol level, but elevated cholesterol esterase may be causative in increasing plasma cholesterol and LDL. Additional experiments further demonstrated that cholesterol esterase can convert the larger and less-atherogenic LDL to the smaller and more atherogenic LDL subspecies in vitro. These results suggest that plasma cholesterol esterase contributes to the formation and accumulation of atherogenic lipoproteins, and thus is a major risk factor for premature atherosclerosis in normal human subjects.