Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Chemical and physical properties.

Subfractions of CLDL (VLDL), Sf 100-400; CLDL2, Sf 60--100; VLDL3, Sf 20--60) and LDL (LDL), Sf 12--20; LDL2, Sf 6--12; LDL3, Sf 3--6) were isolated from the plasma of three normal, three type III and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies were of normal composition but were increased in concentration in the order VLDL1 greater than VLDL2 greater than VLDL3. In the same subjects, although LDL2 was lowered and LDL3 increased, the total plasma LDL concentration was normal. All VLDL subfractions were elevated in the type III group, but in this case VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL1 and was attributed to a substantial alteration in the cholesteryl ester : triglyceride ratio in the particle. A similar argument was used to explain thction in normal or type IV subjects. Particle diameters, determined by laser light-scattering spectroscopy were in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship between the physical and metabolic properties of VLDL and LDL subfractions in type III and IV hyperlipoproteinemia.     

Characterization and metabolic fate of two very-low-density lipoprotein subfractions separated by heparin-sepharose chromatography

Human very-low-density lipoproteins (VLDL) have been separated into two discrete subfractions by heparin-Sepharose chromatography. The retained fraction relative to the unretained fraction is characterized by an increased cholesterol ester/triacylglycerol ratio and an increased ratio of apolipoprotein E relative to apolipoprotein C. We have subfractionated VLDL from type IV hyperlipoproteinemic subjects and characterized these subfractions with respect to (i) composition and (ii) the metabolic fate of apolipoprotein B of each subfraction. The unretained fraction accounted for an average of 42% of total VLDL in type IV subjects. A similar distribution was obtained with VLDL from Type III subjects; however, only 25% of normal VLDL is in the unretained fraction. The apolipoprotein E/apolipoprotein C ratio was 2-8-fold higher in the retained fraction. The distribution of apolipoprotein E isomorphs and the individual C apolipoproteins were similar in each fraction. Retained and unretained fractions were labelled with 125I and/or 131I and injected simultaneously into miniature pigs. Apolipoprotein B of retained fractions was catabolized at a greater rate (fractional catabolic rate = 0.98 h-1 vs. 0.54 h-1, n = 7, P less than 0.05) compared to unretained fractions. These results are consistent with the concept that reduced content of C apolipoproteins in VLDL is correlated with enhanced uptake by perfused rat livers. Apolipoprotein B from retained fractions was converted to intermediate-density lipoproteins (IDL) at a greater rate, and apolipoprotein B from both fractions were converted to low-density lipoproteins (LDL). Although the unretained fraction may be the precursor of the retained fraction, the possibility exists that each fraction is largely synthesized and catabolized independently.     

Lipoprotein lipase- and hepatic triglyceride lipase- promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism

Apolipoprotein E (apoE) is the primary recognition signal on triglyceride-rich lipoproteins responsible for interacting with low density lipoprotein (LDL) receptors and LDL receptor-related protein (LRP). It has been shown that lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) promote receptor-mediated uptake and degradation of very low density lipoproteins (VLDL) and remnant particles, possibly by directly binding to lipoprotein receptors. In this study we have investigated the requirement for apoE in lipase-stimulated VLDL degradation. We compared binding and degradation of normal and apoE-depleted human VLDL and apoE knockout mouse VLDL in human foreskin fibroblasts. Surface binding at 37 degrees C of apoE knockout VLDL was greater than that of normal VLDL by 3- and 40-fold, respectively, in the presence of LPL and HTGL. In spite of the greater stimulation of surface binding, lipase-stimulated degradation of apoE knockout mouse VLDL was significantly lower than that of normal VLDL (30, 30, and 80%, respectively, for control, LPL, and HTGL treatments). In the presence of LPL and HTGL, surface binding of apoE-depleted human VLDL was, respectively, 40 and 200% of normal VLDL whereas degradation was, respectively, 25 and 50% of normal VLDL. LPL and HTGL stimulated degradation of normal VLDL in a dose-dependent manner and by a LDL receptor-mediated pathway. Maximum stimulation (4-fold) was seen in the presence LPL (1 microgram/ml) or HTGL (3 microgram/ml) in lovastatin-treated cells. On the other hand, degradation of apoE-depleted VLDL was not significantly increased by the presence of lipases even in lovastatin-treated cells. Surface binding of apoE-depleted VLDL to metabolically inactive cells at 4 degrees C was higher in control and HTGL-treated cells, but unchanged in the presence of LPL. Degradation of prebound apoE-depleted VLDL was only 35% as efficient as that of normal VLDL. Surface binding of apoE knockout or apoE-depleted VLDL was to heparin sulfate proteoglycans because it was completely abolished by heparinase treatment. However, apoE appears to be a primary determinant for receptor-mediated VLDL degradation.Our studies suggest that overexpression of LPL or HTGL may not protect against lipoprotein accumulation seen in apoE deficiency.     

Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase

In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol

Human VLDL, LDL and HDL (very-low-, low- and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.     

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible.The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.     

Effect of Oral Coenzyme Q10 Supplementation on the Oxidation Resistance of Human VLDL+LDL Fraction: Absorption and Antioxidative Properties of Oil and Granule-Based Preparations

Coenzyme Q10 (Q10) is supposed to be an important endogenous lipid-soluble antioxidant. We studied 60 healthy 46 ± 7 (mean ± SD)-year-old smoking men. They were randomized into three groups to receive oil-based or granular Q10 (90 mg/d) or placebo for 2 months. Oil-based capsule elevated Q10 in plasma by 178% and in VLDL+LDL fraction by 160%. The granular preparation increased Q10 in plasma by 168% and in VLDL+LDL by 127%. However, the 2-month Q10 supplementation did not increase the oxidation resistance of VLDL+LDL fraction, as assessed by copper induced VLDL+LDL oxidation, haemin+H₂O₂-induced VLDL+LDL oxidation, total antioxidative capacity of LDL, and plasma malondialdehyde measurements. The first and the last dose was used to carry out a 12 h pharmacokinetic study (five subjects per group), which indicated that only a small part of supplemented Q10 was absorbed to the circulation in 12 h and that the absorption varied extensively between subjects. Our results suggest that at least among smoking men, 90 mg of orally supplemented Q10 daily does not increase the oxidation resistance of VLDL+LDL. Bioavailability of both the granular and the oil-based Q10 preparation was similar during the long-term supplementation, but one dose of 30 mg had only a marginal effect on the plasma levels of Q10. © 1997 Elsevier Science Inc.     

Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions

Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Interaction of cholesterol ester and triglyceride in human plasma very low density lipoprotein.

The properties of human plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and their extracted lipids were compared using calorimetric, X-ray scattering, and polarizing microscopy techniques. Intact LDL, and cholesterol esters isolated from LDL and VLDL each undergo reversible changes in their physical state around body temperature. These transitions are associated with ordered liquid crystalline to liquid phase changes of the cholesterol esters. In contrast to LDL, VLDL has no reversible transitions and shows no evidence of ordered liquid crystalline structures between 10 and 45 degrees C. Therefore, unlike LDL, VLDL does not contain a separate cholesterol ester region capable of undergoing cooperative melting. Solubility studies at 37 degrees C of cholesterol esters and triglyceride isolated from VLDL show that even at a weight ratio of 1:1, which greatly exceeds the relative amount of cholesterol esters in VLDL, cholesterol ester is completely soluble in triglyceride. Thus, the cholesterol ester in VLDL is not sequestered in a separate domain within VLDL, but is dissolved in the liquid core of the particle.     

Hepatic triglyceride lipase promotes low density lipoprotein receptor-mediated catabolism of very low density lipoproteins in vitro.

We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time.We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.     

Postprandial chylomicrons: potent vehicles for transporting cholesterol from endogenous LDL+HDL and cell membranes to the liver via LCAT and CETP

We examined whether postprandial (PP) chylomicrons (CMs) can serve as vehicles for transporting cholesterol from endogenous cholesterol-rich lipoprotein (LDL+HDL) fractions and cell membranes to the liver via lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities. During incubation of fresh fasting and PP plasma containing [(3)H]cholesteryl ester (CE)-labeled LDL+HDL, both CMs and VLDL served as acceptors of [(3)H]CE or cholesterol from LDL+HDL. The presence of CMs in PP plasma suppressed the ability of VLDL to accept [(3)H]CE from LDL+HDL. In reconstituted plasma containing an equivalent amount of triglycerides from isolated VLDL or CMs, a CM particle was about 40 times more potent than a VLDL particle in accepting [(3)H]CE or cholesterol from LDL+HDLs. When incubated with red blood cells (RBCs) as a source for cell membrane cholesterol, the cholesterol content of CMs, VLDL, LDL, and HDL in PP plasma increased by 485%, 74%, 13%, and 30%, respectively, via LCAT and CETP activities. The presence of CMs in plasma suppressed the ability of endogenous lipoproteins to accept cholesterol from RBCs. Our data suggest that PP CMs may play an important role in promoting reverse cholesterol transport in vivo by serving as the preferred ultimate vehicle for transporting cholesterol released from cell membranes to the liver via LCAT and CETP.     

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.4 x 10(-9) cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 x 10(-8) cm2/s). At 45 degrees C, DT for LDL increases to 1.1 x 10(-8) cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 degrees C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, delta sigma, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P NMR line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 degrees C, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and greater than 120 ppm (HDL3) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein particle size. For analytical purposes, each scan was divided into 12 fractions, representing 12 particle size ranges. The relationship between the estimated cholesterol concentrations in the summed GGE/densitometric fractions corresponding to very low-density lipoproteins (VLDL) + low-density lipoproteins (LDL) and those corresponding to high-density lipoproteins (HDL) and concentrations measured by the heparin-Mn2+ precipitation/enzymatic procedure was linear over a broad range. However, a systematic overestimation of HDL cholesterol concentration and an underestimation of VLDL + LDL cholesterol concentration was apparent. Therefore, correction factors were developed for adjusting the estimates of VLDL + LDL and HDL cholesterol concentrations obtained by the GGE/densitometric method. This analytical method is rapid, repeatable, economical, and useful for genetic and dietary research in which cholesterol concentrations in multiple particle size ranges of lipoproteins must be measured in large numbers of samples. It also is adaptable to immunoblotting procedures for detecting the distribution of specific apolipoproteins among the size-resolved lipoproteins.