Abnormalities of low density lipoproteins in normolipidemic type II diabetic and nondiabetic patients with coronary artery disease.

The characteristics of low density lipoproteins (LDL) of ten non-insulin-dependent diabetic (NIDDM) and ten nondiabetic patients with coronary artery disease (CAD) were investigated and compared to LDL of ten NIDDM patients without CAD and ten healthy persons. All subjects had LDL cholesterol below 160 mg/dl and serum triglycerides below 200 mg/dl. The mean LDL particle size and particle distribution profiles were analyzed by using nondenaturing polyacrylamide gradient gel electrophoresis. The LDL composition and hydrated density distribution were investigated by using density gradient ultracentrifugation. Both NIDDM and nondiabetic CAD patients tended to have larger LDL particles than NIDDM patients without CAD and healthy subjects. The increase of LDL particle size of CAD patients was due to marked enrichment of triglycerides (TG) in their LDL. The percentage content of TG in LDL of NIDDM patients with CAD was 14.5% and in LDL of nondiabetic CAD patients 13.4% compared with 7.9% in LDL of NIDDM patients without CAD and 7.2% in normal-LDL (P less than 0.05 or less between either CAD group and NIDDM without CAD or normals). The LDL TG/apolipoprotein (apo) B weight ratio was significantly higher in both CAD groups compared with LDL of the two groups without CAD (0.70 and 0.68 vs. 0.38 and 0.34, respectively, P less than 0.05, P less than 0.05 and P less than 0.01, P less than 0.01). The LDL total lipid to apoB weight ratio was similar in all four groups. Consistent with this, the hydrated density distributions of LDL in the four groups were similar, the average peak densities being 1.0346 g/ml, 1.0331 g/ml, 1.0331 g/ml, and 1.0331 g/ml, respectively. The findings of this study demonstrate that normolipidemic patients with CAD may have marked abnormalities in th eir LDL composition and these anomalies are present in both diabetic and nondiabetic patients.     

Sialic acid content of low density lipoprotein and its relation to lipid concentrations and metabolism of low density lipoprotein and cholesterol

A low sialic acid content in low density lipoprotein (LDL) has been associated with atherogenicity and coronary artery disease (CAD) in many but not all studies. We investigated associations of the sialic acid-to-apolipoprotein B (apoB) ratio of LDL with lipoprotein lipid concentrations, kinetics of LDL, metabolism of cholesterol, and the presence of CAD in 98 subjects (CAD(+), n = 56; CAD(-), n = 42). The sialic acid ratios of total, dense, and very dense LDL were lower in the CAD(+) than CAD(-) subjects, especially at high sialic acid ratios. The LDL sialic acid ratio was inversely associated with respective lipid and apoB concentrations and positively with lipid-to-apoB ratios of LDL. The transport rates (TRs) for total and dense LDL apoB were negatively associated with their sialic acid ratios. The sialic acid ratio of dense LDL, but not that of total LDL, was inversely correlated with serum levels of cholesterol precursor sterols, indicators of cholesterol synthesis, and positively with serum levels of plant sterols, indicators of cholesterol absorption. In addition, the TR for dense LDL was positively correlated with cholesterol synthesis.In conclusion, a low LDL sialic acid ratio was associated with CAD, high numbers of small LDL particles, and a high TR for LDL apoB, and in dense LDL also with high synthesis and low absorption of cholesterol.     

Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production

We investigated the metabolism of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) apolipoprotein B (apoB) in seven patients with combined hyperlipidemia (CHL), using 125I-labeled VLDL and 131I-labeled LDL and compartmental modeling, before and during lovastatin treatment. Lovastatin therapy significantly reduced plasma levels of LDL cholesterol (142 vs 93 mg/dl, P less than 0.0005) and apoB (1328 vs 797 micrograms/ml, P less than 0.001). Before treatment, CHL patients had high production rates (PR) of LDL apoB. Three-fourths of this LDL apoB flux was derived from sources other than circulating VLDL and was, therefore, defined as "cold" LDL apoB flux. Compared to baseline, treatment with lovastatin was associated with a significant reduction in the total rate of entry of apoB-containing lipoproteins into plasma in all seven CHL subjects (40.7 vs. 25.7 mg/kg.day, P less than 0.003). This reduction was associated with a fall in total LDL apoB PR and in "cold" LDL apoB PR in six out of seven CHL subjects. VLDL apoB PR fell in five out of seven CHL subjects. Treatment with lovastatin did not significantly alter VLDL apoB conversion to LDL apoB or LDL apoB fractional catabolic rate (FCR) in CHL patients. In three patients with familial hypercholesterolemia who were studied for comparison, lovastatin treatment increased LDL apoB FCR but did not consistently alter LDL apoB PR. We conclude that lovastatin lowers LDL cholesterol and apoB concentrations in CHL patients by reducing the rate of entry of apoB-containing lipoproteins into plasma, either as VLDL or as directly secreted LDL.     

Antibodies against modified low-density lipoproteins and their complexes in blood of patients with various manifestations of atherosclerosis

The study included 79 patients with coronary artery disease (CAD), 25 individuals with preclinical atherosclerosis and 59 healthy individuals. Key lipid parameters were examined in all the participants. Levels of antibodies (Abs) (IgG and IgM) against low density lipoproteins (LDL) modified by malondialdehyde (MDA), acetic anhydride and hypochlorite, were determined by the enzyme-linked immunosorbent assay (ELISA). Abs specificity was tested by competitive ELISA. Circulating immune complexes (CIC) were isolated by polyethylene glycol precipitation followed by determination of their cholesterol by the enzymatic method. Abs to hypochlorite-modified LDL (hypochlorite-LDL) detected in the serum samples did not demonstrate cross-reactivity with MDA-modified LDL (MDA-LDL) and acetylated LDL (acetyl-LDL). Patients with CAD had increased levels of CIC (p < 0.0001) and decreased levels of Abs (IgM) to hypochlorite- LDL, compared with healthy controls and patients with preclinical atherosclerosis (p = 0.006). A correlation between the levels of Abs (IgG) to the hypochlorite-LDL and Abs to MDA-LDL and acetyl-LDL was found. The content of the Abs (IgM) to MDA-LDL and acetyl-LDL correlated with CIC-cholesterol concentrations, while lipid parameters did not correlate with Abs levels.     

Differences in carbohydrate content of low density lipoproteins associated with low density lipoprotein subclass patterns

The neutral carbohydrate content of both the protein (apoB) and lipid fractions of low density lipoproteins (LDL) from subjects with a predominance of small, dense LDL (subclass pattern B) was found to be lower than in subjects with larger LDL (subclass pattern A): 45 +/- 12 versus 64 +/- 13 mg/g apoLDL, and 58 +/- 8 versus 71 +/- 8 mg/g apoLDL (P less than 0.0005 for both). Sialic acid content of LDL lipids, but not apoB, was also reduced in subclass pattern B. ApoB and glycolipid carbohydrate content of total LDL and LDL density subfractions declined with increasing LDL density and decreasing particle diameter. Moreover, in LDL subfractions from pattern B subjects, carbohydrate content of LDL apoB, but not LDL glycolipid, was significantly lower in comparison with particles of similar size from pattern A subjects. Thus, in LDL subclass pattern B, reductions in LDL carbohydrate content are associated both with reduced concentrations of larger carbohydrate-enriched LDL subclasses, and with reduced glycosylation of apoB in all LDL particles. LDL glycolipids may vary with overall lipid content of LDL particles, but variation in apoB glycosylation may indicate differences in pathways for LDL production, and reduced apoB glycosylation may reflect the altered metabolic state responsible for LDL subclass pattern B.     

ApoE and ApoC-I polymorphisms: association of genotype with cardiovascular disease phenotype in African Americans

Apolipoproteins (apo) E and C-I are components of triglyceride (TG)-rich lipoproteins and impact their metabolism. Functional polymorphisms have been established in apoE but not in apoC-I. We studied the relationship between apoE and apoC-I gene polymorphisms and plasma lipoproteins and coronary artery disease (CAD) in 211 African Americans and 306 Caucasians. In African Americans but not in Caucasians, apoC-I H2-carriers had significantly lower total and LDL cholesterol and apoB levels, and higher glucose, insulin, and HOMA-IR levels compared with H1 homozygotes. Differences across CAD phenotypes were seen for the apoC-I polymorphism. African-American H2-carriers without CAD had significantly lower total cholesterol (P < 0.001), LDL cholesterol (P < 0.001), and apoB (P < 0.001) levels compared with H1 homozygotes, whereas no differences were found across apoC-I genotypes for African Americans with CAD. Among African-American apoC-I H1 homozygotes, subjects with CAD had a profile similar to the metabolic syndrome (i.e., higher triglyceride, glucose, and insulin) compared with subjects without CAD. For African-American H2-carriers, subjects with CAD had a pro-atherogenic lipid pattern (i.e., higher LDL cholesterol and apoB levels), compared with subjects without CAD. ApoC-I genotypes showed an ethnically distinct phenotype relationship with regard to CAD and CAD risk factors.     

VAP II analysis of lipoprotein subclasses in mixed hyperlipidemic patients on treatment with ezetimibe/simvastatin and fenofibrate

This analysis evaluates the effects on lipoprotein subfractions and LDL particle size of ezetimibe/simvastatin with or without coadministration of fenofibrate in patients with mixed hyperlipidemia. This multicenter, double-blind, placebo-controlled, parallel-group study included 611 patients aged 18-79 years randomized in 1:3:3:3 ratios to one of four 12 week treatment groups: placebo; ezetimibe/simvastatin 10/20 mg/day; fenofibrate 160 mg/day; or ezetimibe/simvastatin 10/20 mg/day + fenofibrate 160 mg/day. At baseline and study endpoint, cholesterol associated with VLDL, intermediate density lipoprotein (IDL), LDL, and HDL subfractions was quantified using the Vertical Auto Profile II method. LDL particle size was determined using segmented gradient gel electrophoresis. Whereas fenofibrate reduced cholesterol mass within VLDL and IDL, and shifted cholesterol from dense LDL subfractions into the more buoyant subfractions and HDL, ezetimibe/simvastatin reduced cholesterol mass within all apolipoprotein B-containing particles without significantly shifting the LDL particle distribution profile. When administered in combination, the effects of the drugs were complementary, with more-pronounced reductions in VLDL, IDL, and LDL, preferential loss of more-dense LDL subfractions, and increased HDL, although the effects on most lipoprotein subfractions were not additive. Thus, ezetimibe/simvastatin + fenofibrate produced favorable effects on atherogenic lipoprotein subclasses in patients with mixed hyperlipidemia.     

Relationship of IgG and IgM autoantibodies and immune complexes to oxidized LDL with markers of oxidation and inflammation and cardiovascular events: results from the EPIC-Norfolk Study

Levels of IgG and IgM autoantibodies (AA) to malondialdehyde (MDA)-LDL and apoB-immune complexes (ICs) were measured in 748 cases and 1,723 controls in the EPIC-Norfolk cohort and their association to coronary artery disease (CAD) events determined. We evaluated whether AA and IC modify CAD risk associated with secretory phospholipase A(2) (sPLA(2)) type IIA mass and activity, lipoprotein-associated PLA(2) activity, lipoprotein (a) [Lp(a)], oxidized phospholipids on apoB-100 (OxPL/apoB), myeloperoxidase, and high sensitivity C-reactive protein. IgG ICs were higher in cases versus controls (P = 0.02). Elevated levels of IgM AA and IC were inversely associated with Framingham Risk Score and number of metabolic syndrome criteria (p range 0.02-0.001). In regression analyses adjusted for age, smoking, diabetes, LDL-cholesterol, HDL-cholesterol, and systolic blood pressure, the highest tertiles of IgG and IgM AA and IC were not associated with higher risk of CAD events compared with the lowest tertiles. However, elevated levels of IgM IC reduced the risk of Lp(a) (P = 0.006) and elevated IgG MDA-LDL potentiated the risk of sPLA(2) mass (P = 0.018). This epidemiological cohort of initially healthy subjects shows that IgG and IgM AA and IC are not independent predictors of CAD events but may modify CAD risk associated with elevated levels of oxidative biomarkers.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

Influence of LDL apheresis on LDL subtypes in patients with coronary heart disease and severe hyperlipoproteinemia

Epidemiologic studies and in vitro experiments indicate that low density lipoprotein (LDL) subtypes differ concerning their atherogenic potential. Small, dense LDL are more atherogenic than large, buoyant LDL. LDL apheresis is a potent therapeutic modality to lower elevated LDL-cholesterol. It is unknown whether such therapy induces a shift in the LDL subtype distribution. In this study we evaluated the influence of LDL apheresis on the LDL subtype distribution in patients with CHD and familial hypercholesterolemia (FH, n = 22), combined hyperlipidemia (CHLP, n = 6), or Lp[a]-hyperlipoproteinemia (Lp[a]-HLP, n = 4) regularly treated by LDL apheresis (immunoadsorption (n = 14), HELP apheresis (n = 8), dextran sulfate adsorption (n = 7), cascade filtration (n = 3)). On the basis of 6 LDL subfractions (d 1.020;-1.057 g/mL) isolated by density gradient ultracentrifugation the LDL-density profile was determined in each patient before and after apheresis. There was a relative increase of LDL-subfractions 1, 2, and 3 (P < 0.01, P < 0. 05, and P < 0.01, respectively) and a concomitant decrease of LDL subfractions 5 and 6 (P < 0.05) after apheresis. Subgroup analysis indicates that the degree of the small, dense LDL reduction was much more prominent in patients with CHLP compared to patients with FH or Lp[a]-HLP, whereas the type of apheresis technique had no effect. The extent of small, dense LDL reduction correlated with the preapheresis concentrations of small, dense LDL and triglycerides but not with the extent of triglyceride reduction.We conclude that LDL apheresis not only decreases LDL mass, but also improves LDL-density profile, particularly in patients with CHLP.     

A common genetic mechanism determines plasma apolipoprotein B levels and dense LDL subfraction distribution in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCH) is a common lipid disorder characterized by elevations of plasma cholesterol and/or triglyceride in first-degree relatives. A predominance of small, dense LDL particles and elevated apolipoprotein B (apoB) levels is commonly found in members of FCH families. Many studies have investigated the genetic mechanisms determining individuals' lipid levels, in FCH families. Previously, we demonstrated a major gene effect on LDL particle size and codominant Mendelian inheritance involved in determination of apoB levels in a sample of 40 well-defined FCH families. An elevation of apoB levels is associated metabolically with a predominance of small, dense LDL particles in FCH. To establish whether a common gene regulates both traits, we conducted a bivariate genetic analysis to test the hypothesis of a common genetic mechanism. In this study, we found that 66% of the total phenotypic correlation is due to shared genetic components. Further bivariate segregation analysis suggested that both traits share a common major gene plus individual polygenic components. This common major gene explains 37% of the variance of adjusted LDL particle size and 23% of the variance of adjusted apoB levels. Our study suggests that a major gene that has pleiotropic effects on LDL particle size and apoB levels may be the gene underlying FCH in the families we studied.     

Computed high concentrations of low-density lipoprotein correlate with plaque locations in human coronary arteries

Subendothelial accumulation of low-density lipoprotein (LDL) in arterial walls is an initiator of atherosclerotic plaque formation. We report here on the correlation between healthy state subendothelial LDL concentration distribution and sites of subsequent plaque formation in coronary arteries of patients with coronary artery disease (CAD). We acquired left (LCA) and right coronary artery (RCA) and atherosclerotic plaque geometries of 60 patients with CAD using dual-source computed tomography angiography. After virtually removing all plaques to obtain an approximation of the arteries' healthy state, we calculated LDL concentration in the artery walls as a function of local lumen-side shear stress. We found that maximum subendothelial LDL concentrations at plaque locations were, on average, 45% (RCA) and 187% (LCA) higher than the respective average subendothelial concentration. Our results demonstrate that locally elevated subendothelial LDL concentration correlates with subsequent plaque formation at the same location.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Microsomal triglyceride transfer protein -493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.     

Density distribution of electronegative LDL in normolipemic and hyperlipemic subjects

The density distribution of electronegative LDL [LDL(-)], a cytotoxic and inflammatory fraction of LDL present in plasma, was studied in 10 normolipemic (NL), 6 FH, and 11 hypertriglyceridemic (HTG) subjects. Six LDL subclasses of increased density (LDL1 to LDL6) were isolated by density-gradient ultracentrifugation (DGU). NL and FH subjects showed prevalence of light LDL, whereas HTG subjects showed prevalence of dense LDL. LDL(-) proportion was determined from total LDL or LDL-density subclasses by anion-exchange chromatography. LDL from FH patients had increased LDL(-) (35.1 +/- 9.9%) compared with LDL from NL and HTG subjects (9.4 +/- 2.3% and 12.3 +/- 4.3%, respectively). Most LDL(-) was contained in dense subclasses in NL (LDL4-6, 67.7 +/- 3.1%) whereas most of LDL(-) from FH patients were contained in light LDL subclasses (LDL1-3) (86.2 +/- 1.6%). In these subjects, simvastatin therapy decreased LDL(-) to 28.2 +/- 6.7% and 21.2 +/- 5.6% at 3 and 6 months of treatment, respectively, due mainly to decreases in light LDL subclasses. In HTG subjects, half LDL(-) was contained in dense LDL subclasses (LDL4-6, 46.1 +/- 2.0%). Non-denaturing acrylamide gradient gel electrophoresis concurred with DGU data, as LDL(-) from NL showed a single band of lower size than non-electronegative LDL [LDL(+)], whereas LDL(-) from FH and HTG presented bands of greater size than its respective LDL(+). These results reveal the existence of light and dense LDL(-), indicate that hyperlipemia could promote the formation of light LDL(-) and suggest that LDL(-) could have different origins.     

Altered particle size distribution of apolipoprotein A-I-containing lipoproteins in subjects with coronary artery disease

Plasma high density lipoproteins (HDL) can be separated into two subpopulations of apolipoprotein A-I-containing particles: those that also contain apoA-II [Lp(AI w AII)] and those that do not [Lp(AI w/o AII)]. These particles were isolated by immunoaffinity chromatography from 17 men (9 normolipidemic (NL), 8 hyperlipidemic (HL) with symptomatic coronary artery disease (CAD), from 17 NL men without any symptoms of CAD (healthy controls), and from 10 NL men with entirely normal coronary arteriograms (CAD-free controls). The distributions of particle size in these two subpopulations were determined by gradient gel electrophoresis and densitometric scanning. Approximately half of the Lp(AI w AII) particles in all subjects were distributed in the 8.2-9.2 nm interval. For patients with CAD, a greater fraction of the particles were small, in the 7.0-8.2 nm interval [33% in CAD vs. 26% in CAD-free controls (P less than 0.01) and 19% in healthy controls (P less than 0.0001)], and a smaller fraction of the particles were in the 9.2-11.2 nm interval (14% in CAD vs. 24% in CAD-free control (P less than 0.002) and healthy control groups (P less than 0.001). The Lp(AI w/o AII) of both control groups were primarily composed of two discrete subpopulations in the 8.2-9.2 nm and the 9.2-11.2 nm intervals. In CAD patients there were fewer particles in the 9.2-11.2 nm size interval (23% in CAD vs. 33% in CAD-free controls (P less than 0.005) and 36% in healthy controls (P less than 0.0001), and more particles in the smallest 7.0-8.2 nm size interval (32% in CAD vs. 23% in CAD-free controls (P less than 0.01) and 18% in healthy controls (P less than 0.001]. Thus, the spectrum of HDL particle sizes in patients with CAD tends to be shifted toward the smaller particle when compared with the two control groups. This was observed in both NL and HL patients with HDL cholesterol (CH) values in the normal range. As a group, CAD patients had lower HDL (42 +/- 7 mg/dl) and HDL2 (6 +/- 4 mg/dl) CH than healthy (HDL: 49 +/- 7, HDL2: 12 +/- 6 mg/dl) and CAD-free (HDL: 51 +/- 9, HDL2: 12 +/- 6 mg/dl) controls. When controls and patients were compared for their frequencies of abnormal HDL CH levels and particle sizes, abnormalities in HDL and HDL2 CH levels were not significantly more frequent (twofold) among CAD patients than among controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immuno-electron cryo-microscopy imaging reveals a looped topology of apoB at the surface of human LDL

A single copy of apoB is the sole protein component of human LDL. ApoB is crucial for LDL particle stabilization and is the ligand for LDL receptor, through which cholesterol is delivered to cells. Dysregulation of the pathways of LDL metabolism is well documented in the pathophysiology of atherosclerosis. However, an understanding of the structure of LDL and apoB underlying these biological processes remains limited. In this study, we derived a 22 Å-resolution three-dimensional (3D) density map of LDL using cryo-electron microscopy and image reconstruction, which showed a backbone of high-density regions that encircle the LDL particle. Additional high-density belts complemented this backbone high density to enclose the edge of the LDL particle. Image reconstructions of monoclonal antibody-labeled LDL located six epitopes in five putative domains of apoB in 3D. Epitopes in the LDL receptor binding domain were located on one side of the LDL particle, and epitopes in the N-terminal and C-terminal domains of apoB were in close proximity at the front side of the particle. Such image information revealed a looped topology of apoB on the LDL surface and demonstrated the active role of apoB in maintaining the shape of the LDL particle.     

Analysis and quantitation of biotinylated apoB-containing lipoproteins with streptavidin-Cy3

Non denaturing gradient gel electrophoresis (GGE) is commonly used to analyze the size distribution of lipoprotein particles. Its relatively low sensitivity and linear dynamic range limit use of GGE to quantify protein content of lipoproteins. We demonstrate a new high sensitivity method for analysis and quantitation of biotinylated apolipoprotein B (apoB)-containing lipoproteins using a fluorescent streptavidin-Cy3 conjugate and non covalent preelectrophoretic binding. Forty-four lipoprotein subfractions spanning the VLDL and LDL particle spectrum subfractions (11 each from four human subjects) were prepared by density gradient ultracentrifugation. An aliquot of each sample was biotinylated and GGE was performed. Gels also were stained for lipid with Oil Red O (32 samples) and for protein with Coomassie Brilliant Blue (30 samples). There was a significant relationship between the Cy3 fluorescent label area under the curve and the mass of apoB (P < 0.02-0.004) and total cholesterol (P < 0.03-0.004). Particle diameters of each absorbence/fluorescent peak were comparable between Oil-Red O and streptavidin-Cy3 treated biotinylated lipoproteins (+/-3.54 A, P = 0.3). Biotinylation and prestaining of lipoprotein particle with streptavidin-Cy3 provides a new fluorescence-based method for detection and quantitative analysis of lipoprotein subspecies by gradient gel electrophoresis.     

Solubilization of low-density lipoprotein with sodium deoxycholate and recombination of apoprotein B with dimyristoylphosphatidylcholine

Apoprotein B (apoB) of human plasma low-density lipoprotein (LDL) (d 1.025-1.050 g/mL) has been solubilized with solid sodium deoxycholate (NaDC) above its critical micellar concentration. ApoB is isolated by gel-filtration chromatography as a mixed micellar complex of protein and detergent in high yield in a lipid-free form. A soluble apoB-dimyristoylphosphatidylcholine (DMPC) complex has been prepared by incubation of aqueous solutions of apoB-NaDC and DMPC-NaDC (2/1 w/w) at room temperature with detergent removal by extensive dialysis. A combination of gel chromatographic and density gradient fractionation of DMPC-apoB incubation mixtures demonstrates that a reasonably well-defined complex of DMPC and apoB is formed with a 4:1 w/w lipid:protein ratio. Negative-stain electron microscopy shows these particles to be single-bilayer phospholipid vesicles with a diameter of 210 +/- 20 A into which the apoB is incorporated. Circular dichroic spectra of NaDC-solubilized apoB show apoB to have similar conformation to that seen in the native LDL particle. However, apoB that has been complexed with DMPC exhibits more alpha-helix. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band (apparent Mr 366000) for apoB after solubilization, purification, and interaction with phospholipid. The behavior of apoB during its reassociation with phospholipid and the structural features of the DMPC-apoB particle are similar to those observed in the interaction of solubilized membrane proteins with lipid rather than that of other apo-lipoproteins.