Catabolism of very low density lipoproteins in the rabbit. Effect of changing composition and pool size.

To determine the metabolic mechanism of hypercholesterolemia in rabbits produced by feeding cholesterol-rich diets, control and hypercholesterolemic rabbits were injected with I-labelled very low density lipoproteins (VLDL, d 1.006 g/ml) from control and/or hypercholesterolemic donors. Apolipoprotein B in VLDL decayed biphasically. The first phase occurred much more rapid than the second. 95% of the VLDL apolipoprotein B was catabolized via the first phase (t1/2 = 0.55 +/- 0.19 h) in normal rabbit with the immediate appearance of this radioactivity in intermediate density lipoproteins (IDL, d 1.006-1.025 g/ml) and low density lipoproteins (LDL, d 1.025-1.063 g/ml). The apolipoproteins C and E at the same time were transferred to high density lipoproteins where they decayed biphasically. The apolipoprotein B from hypercholesterolemic VLDL in the normal recipient disappeared at a similar rate as from normal VLDL via phase I; however, it was incompletely converted to IDL and LDL. Apolipoprotein B from normal VLDL in cholesterol-fed rabbits disappeared at a normal rate via phase I, but only 82% was catabolized by this phase. Hypercholesterolemic VLDL injected into the hypercholesterolemic recipient was less rapidly catabolized via phase I (T1/2 = 2.5 +/- 0.89 H) and only a small fraction was converted to IDL and LDL.     

Isolation and characterization of an apoA-II-containing lipoprotein (LP-A-II:B complex) from plasma very low density lipoproteins of patients with Tangier disease and type V hyperlipoproteinemia

Previous studies have shown that very low density lipoproteins (VLDL) from patients with Tangier disease are less effective as a substrate for human milk lipoprotein lipase (LPL) than VLDL from normal controls as assessed by measuring the first order rate constant (k1) of triglyceride hydrolysis. Tangier VLDL also has a higher content of apolipoprotein (apo) A-II than normal VLDL. To explore the possible relationship between the relatively high concentration of apoA-II in VLDL and low k1 values, Tangier VLDL were fractionated on an anti-apoA-II immunosorber. The retained fraction contained a newly identified triglyceride-rich lipoprotein characterized by the presence of apolipoproteins A-II, B, C-I, C-II, C-III, D, and E (LP-A-II:B:C:D:E or LP-A-II:B complex), whereas the unretained fraction consisted of previously identified triglyceride-rich apoB-containing lipoproteins free of apoA-II. In VLDL from patients with Tangier disease or type V hyperlipoproteinemia, the LP-A-II:B complex accounted for 70-90% and 25-70% of the total apoB content, respectively. The LP-A-II:B complexes had similar lipid and apolipoprotein composition; they were poor substrates for LPL as indicated by their low k1 values (0.014-0.016 min-1). In contrast, the apoA-II-free lipoproteins present in unretained fractions were effective substrates for LPL with k1 values equal to or greater than 0.0313 min-1. These results indicate that triglyceride-rich lipoproteins consist of several apoB-containing lipoproteins, including the LP-A-II:B complex, and that lipoprotein particles of similar size and density but distinct apolipoprotein composition also possess distinct metabolic properties.     

Identification and partial characterization of discrete apolipoprotein A-containing lipoprotein particles secreted by human hepatoma cell line HepG2

The purpose of this study was to identify the apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. The apolipoprotein A-containing lipoproteins separated from apolipoprotein B-containing lipoproteins by affinity chromatography of culture medium on concanavalin A were fractionated on an immunosorber with monoclonal antibodies to apolipoprotein A-II. The retained fraction contained apolipoproteins A-I, A-II and E, while the unretained fraction contained apolipoproteins A-I and E. Both fractions were characterized by free cholesterol as the major and triglycerides and cholesterol esters as the minor neutral lipids. Further chromatography of both fractions on an immunosorber with monoclonal antibodies to apolipoprotein A-I showed that 1) apolipoprotein A-II only occurs in association with apolipoprotein A-I, 2) apolipoprotein A-IV is only present as part of a separate lipoprotein family (lipoprotein A-IV), and 3) apolipoprotein E-enriched lipoprotein A-I:A-II and lipoprotein A-I are the main apolipoprotein A-containing lipoproteins secreted by HepG2 cells.     

Retention of apolipoprotein B and cholesterol by perfused heart during lipolysis of very-low-density lipoprotein

The fate and mechanism of removal of apolipoproteins and lipids of human very-low-density lipoproteins were determined in the perfused rat heart. Approx. 50% of the VLDL triacylglycerol was hydrolyzed during a 2 h perfusion. Phospholipid phosphorus, apolipoproteins C-II, C-III and E were quantitatively recovered in the medium. However, there was a loss of unesterified (17 +/- 6%) and esterified (19 +/- 8%) cholesterol from the perfusion medium. Apolipoprotein B was retained by the heart, as determined by the loss of immunoassayable apolipoprotein B (30 +/- 5%) or the uptake of 125I-labelled apolipoprotein of VLDL (9 +/- 2%) from the perfusion medium. The discrepancy in the two methods for estimating apolipoprotein removal was shown to be due to the modification of apolipoprotein B-containing lipoproteins, which was such that they were no longer precipitated with antibodies to apolipoprotein B. The labelled apolipoprotein B, retained by the heart, could be partially released by perfusion of the heart with buffer containing heparin (14 +/- 2%) or trypsin (50 +/- 2%). Labelled apolipoprotein uptake by the heart was reduced by 90% when lipoprotein lipase was first released by heparin or when VLDL was treated with 1,2-cyclohexanedione to modify arginine residues of apolipoproteins. Very little extensive degradation of the apoprotein to low molecular weight material occurred during the 2 h perfusion, since 95% of the tissue label was precipitated by trichloroacetic acid. It is concluded that there is retention of apolipoprotein B, cholesteryl ester and cholesterol by the perfused heart during catabolism of VLDL. The data are consistent with the concept that the retention of apolipoprotein B requires membrane-bound lipoprotein lipase or an interaction with the cell surfaces that is modified by heparin. The overall process also involves arginine residues of apolipoproteins. At least 50% of the labelled apolipoprotein retained in the tissue is associated with lipoprotein lipase and other cell surface sites, while the remainder may be taken up by the cells.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.     

Inhibitory effects of C apolipoproteins from rats and humans on the uptake of triglyceride-rich lipoproteins and their remnants by the perfused rat liver

Like rat C apolipoproteins, each of the C apolipoproteins from human blood plasma (C-I, C-II, C-III-1, and C-III-2) bound to small chylomicrons from mesenteric lymph of estradiol-treated rats and inhibited their uptake by the isolated perfused rat liver. This inhibitory effect of the C apolipoproteins was independent of apolipoprotein E, which is present only in trace amounts in these chylomicrons. Addition of rat apolipoprotein E to small chylomicrons from mesenteric lymph of normal rats did not displace C apolipoproteins and had no effect on the uptake of these particles by the perfused liver, indicating that an increased ratio of E apolipoproteins to C apolipoproteins on chylomicron particles, unaccompanied by depletion of the latter, may not promote recognition by the chylomicron remnant receptor. The hepatic uptake of remnants of rat hepatic very low density lipoproteins (VLDL) and small chylomicrons, which had been produced in functionally eviscerated rats, was also inhibited by addition of C apolipoproteins. These observations are consistent with the hypothesis that the addition of all of the C apolipoproteins to newly secreted chylomicrons and VLDL inhibits premature uptake of these particles by the liver and that depletion of all of these apolipoproteins from remnant particles facilitates their hepatic uptake. Remnants of chylomicrons and VLDL incubated with rat C apolipoproteins efficiently took up C-III apolipoproteins, but not apolipoprotein C-II (the activator protein for lipoprotein lipase). Preferential loss of apolipoprotein C-II during remnant formation may regulate the termination of triglyceride hydrolysis prior to complete removal of triglycerides from chylomicrons and VLDL.     

Post-secretory acquisition of apolipoprotein E by nascent rat hepatic very-low-density lipoproteins in the absence of cholesteryl ester transfer

Previous work has shown that nascent hepatic very-low-density lipoproteins (VLDL) in the rat are biosynthesized without the obligatory co-factor (apolipoprotein C-II) for lipoprotein lipase-mediated hydrolysis of their core triacylglycerols. Upon secretion, apolipoproteins C-II and C-III are rapidly transferred to the particles from high-density lipoprotein (HDL) within the space of Disse and upon the entry into the plasma. Here we extend those studies to include observations on the apolipoprotein E content and lipid composition of nascent hepatic VLDL before and after exposure to plasma components. We have elected to use hepatic secretory vesicle VLDL rather than liver perfusate VLDL as truly representative of the nascent lipoproteins. Nascent VLDL from fed rats has an apolipoprotein B/E ratio of 6.6 ± 0.5, whereas that from fasted animals is 13.9 ± 2.3. Incubation of nascent VLDL from fed and fasted rats with d > 1.063 g/ml rat serum, HDL or the d > 1.21 g/ml fraction resulted in a mass transfer of apoliproprotein E to the VLDL such that the apolipoprotein B/E ratio decreased to at least that of serum VLDL (3.4 ± 0.3). The d > 1.21 g/ml fraction appeared to contain a species of apolipoprotein E which most actively transferred to VLDL. The acquisition of apolipoprotein E by nascent secretory vesicle VLDL was attended by a loss of phospholipids, particularly the C₄₀ (stearoylarachidonyl) molecular species, and an increase in the cholesterol-to-phospholipid ratio from 0.11 ± 0.01 to 0.18 ± 0.03. No evidence was obtained to suggest a simultaneous acquisition of cholesteryl esters upon incubation of nascent VLDL with VLDL-free serum. We conclude that nascent hepatic VLDL is modified after secretion by acquisition of apolipoproteins C-II, C-III and E with a concomitant loss of phospholipids.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.     

Changes in plasma very low density and low density lipoprotein content, composition, and size after a fatty meal in normo- and hypertriglyceridemic man.

Four subfractions of plasma VLDL characterized by decreasing Sf value and LDL were isolated by density gradient preparative ultracentrifugation from normotriglyceridemic (NTG) and hypertriglyceridemic (HTG) (type IV) subjects in the fasting state and after a fatty meal. Chemical analysis and computation of numbers of particles in each fraction showed that the hyperlipidemia of type IV subjects was accounted for by an increase in total numbers of VLDL and a shift in the distribution of VLDL towards particles of larger diameter. Postprandial hyperlipidemia was due to the presence of chylomicron remnants rather than intact chylomicrons, and was accounted for by an increase in particle diameter of the largest VLDL subfraction rather than by an increase in particle numbers. Postprandial hyperlipedemia was accompanied by a shift in the distribution of VLDL towards particles of larger diameter in both NTG and HTG subjects, probably because of competition for the triglyceride-depletion process between chylomicrons and hepatic VLDL. Most chylomicron remnants were removed from the circulation without degradation to smaller VLDL or to LDL, but some remnants were sufficienty small to contribute to smaller VLDL subfractions. The LDL of type IV subjects contained more apoprotein B than those from NTG subjects, and this difference was associated with increases in diameter, molecular weight, density, and the ratio of protein: phospholipid in LDL from type IV subjects. Defective degradation of large VLDL to small VLDL, and of VLDL to LDL may be related to this alteration in apoprotein B content of the lipoproteins in type IV subjects.     

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.     

High-density lipoprotein subpopulations as substrates for the transfer of cholesteryl esters to very-low-density lipoproteins.

1. Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoprotein-deficient serum (LPDS) which contained either [3H]cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, B and C) were successively eluted from each preparation of HDL, HDL2 and HDL3. When the labelling was done at 37 degrees C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4 degrees C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 2. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. 3. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37 degrees C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4 degrees C or when human LPDS was replaced with rat LPDS. 4. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent Vmax for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent Km values were very similar, which suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.     

Apolipoprotein and lipid distribution between vesicles and HDL-like particles formed during lipolysis of human very low density lipoproteins by perfused rat heart

A study was undertaken to determine the relative association of lipid and apolipoproteins among lipoproteins produced during lipolysis of very low density lipoproteins (VLDL) in perfused rat heart. Human VLDL was perfused through beating rat hearts along with various combinations of albumin (0.5%), HDL2, the infranatant of d greater than 1.08 g/ml of serum, and labeled sucrose. The products were resolved by gel filtration, ultracentrifugation, and hydroxylapatite chromatography. The composition of the lipoprotein products was assessed by analysis of total lipid profiles by gas-liquid chromatography and immunoassay of apolipoproteins. A vesicle particle, which trapped and retained 1-2% of medium sucrose, co-isolated with VLDL and VLDL remnants by gel filtration chromatography but primarily with the low density lipoprotein (LDL) fraction when isolated by ultracentrifugation. The vesicle was resolved from apoB-containing LDL lipolysis products by hydroxylapatite chromatography of the lipoproteins. The vesicle lipoprotein contained unesterified cholesterol (34%), phosphatidylcholine and sphingomyelin (50%), cholesteryl ester (6%), triacylglycerol (5%), and apolipoprotein (5%). The apolipoprotein consisted of apoC-II (7%), apoC-III (93%), and trace amounts of apoE (1%). When viewed by electron microscopy the vesicles appeared as rouleaux structures with a diameter of 453 A, and a periodicity of 51.7 A. The mass represented by the vesicle particle in terms of the initial amount in VLDL was: cholesterol (5%), phosphatidylcholine and sphingomyelin (3%), apoC-II (0.5%), apoC-III (2.2%). The majority of the apoC and E released from apoB-containing lipoproteins was associated with neutral-lipid core lipoproteins proteins which possessed size characteristics of HDL. The vesicles were also formed in the presence of HDL and serum and were not disrupted by serum HDL. It is concluded that lipolysis of VLDL in vitro results in the production of VLDL remnants and LDL apoB-containing lipoproteins, as well as HDL-like lipoproteins. A vesicular lipoprotein which has many characteristics of lipoprotein X found in cholestasis, lecithin: cholesterol acyltransferase deficiency, and during Intralipid infusion is also formed. The majority of apolipoprotein C and E released from apoB-containing lipoproteins is associated with the HDL-like lipoprotein. It is suggested that the formation and stability of the vesicle lipoprotein may be related to the high ratio of cholesterol/phospholipid in this particle.     

Lipid and apolipoprotein distribution as a function of density in equine plasma lipoprotein

1. Equine lipoproteins were isolated from plasma by density gradient ultracentrifugation and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. VLDL and IDL were present at low concentration (0.2 mg/ml). Two apoB components of Mr corresponding to human apoB-100 and one apoB-48-like component were represented in VLDL fraction. 3. LDL-1 and LDL-2 subfractions have displayed an almost equal concentration (0.4 mg/ml). Two apoB-100-like components were the major apolipoproteins in each fraction. Small amounts of apoB-48-like component were detectable in LDL-1 and LDL-2. 4. HDL-2 represented a major class of equine lipoproteins (1.8 mg/ml). ApoA-1-like component was the dominant protein in HDL-1, HDL-2 and HDL-3. Dimeric apoA-II-like components were slightly represented in HDL subfractions. 5. HDL-3 displayed the same apolipoprotein pattern as HDL-1 and HDL-2, but two further minor proteins of Mr 20,000 and 14,000 were detected. 6. VHDL represented a minor class of lipoprotein (0.2 mg/ml). ApoA-I-like component was the major apolipoprotein of VHDL. Small amounts of apoA-IV-like, apoE-like, and Mr 55,000 protein were detectable. 7. ApoC-like of Mr lower than 10,000 was represented in all equine lipoprotein classes.     

Lipoprotein synthesis and secretion by cultured rat hepatocytes. Parallel inhibition of secretion of VLDL, HDL and albumin by monensin

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)