Presence Of High-density Lipoprotein Research Articles

In Vitro Interaction of Sized and Unsized Liposome Vesicles with High Density Lipo Proteins

An in vitro method of interaction of liposome vesicles with high density lipoproteins (HDL) was studied. A formulation of lipids was hydrated, and homogenized with a high shear mixer and designed as “unsized” liposomes. A portion of this preparation was further processed by passing through a polycarbonate membrane and it is labelled as “sized”. Both these preparations were incubated with HDL. Destabilization rate of liposome preparations in presence of HDL was determined. Time course of drug release from sized and unsized liposomes was followed upon HDL addition to liposomes. These in vitro studies clearly showed that HDL destabilized the small sized liposomes (0.25 ± 0.09 Hm). The destabilization effect of HDL on unsized is not significant.

Fate of fatty acids taken up as cholesteryl ester by rat hepatocytes in primary culture from high-density lipoprotein

Primary culture of rat hepatocytes were incubated in the presence of high-density lipoproteins (HDL) labelled with [1- 14C]oleyl or [1- 14C]linoleyl cholesteryl ester. Labelled HDL were prepared by selective delipidation with heptane, relipidation and sequential ultracentrifugations. Hepatocytes took up cholesteryl esters and cholesteryl ether their non-hydrolizable analog, at the same rate. The uptake increased with time, the cholesteryl ester/protein ratio and the amount of added HDL. It was not dependent on the nature of acyl chain or on the nature of the bond. The uptake did not depend on a specific interaction between HDL and cell membranes, since cholesteryl ester was taken up from HDL to the same extent as from albumin complexes. Linoleic and oleic acids released from cholesteryl esters taken up by hepatocytes were mainly reesterified into phosphatidylcholine and triacylglycerols. Linoleic acid was preferentially channelled into PC. A portion of these lipids were secreted by hepatocytes during a 24-h reincubation in a medium devoid of lipoprotein. Nearly the same amount of radioactivity was recovered in secreted phospholipids as in secreted triacylglycerols, in contrast with hepatocytes labelled with free fatty acids which secreted very little radioactivity as phospholipids. From these results and the high content in polyunsaturated fatty acids of cholesteryl esters, one can hypothesize that hepatic cholesteryl ester uptake may contribute to biliary phosphatidylcholine production, and therefore to polyunsaturated fatty acid sparing.

Uptake and utilization of double-labeled high-density lipoprotein sphingomyelin in isolated brain capillaries of adult rats.

Isolated rat brain capillaries were incubated in the presence of high-density lipoprotein (HDL) containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin. This double-labeled sphingomyelin was taken up in a concentration-dependent manner. Cerebral capillary-associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hydrolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of incorporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of "pulse-chase" experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidyl-choline of capillaries.

Promotion of Lymphocyte Growth by High Density Lipoproteins (HDL): Physiological Significance of the HDL Binding Site

The characteristics and physiological relevance of the high density lipoprotein (HDL) binding site on unstimulated and mitogen activated human peripheral blood lymphocytes have been investigated. At 37 °C, specific binding/uptake of fluorescent (dioctadecylin-docarbocyanine, DiI) HDL was observed by cells from healthy donors as well as by those from low density lipoprotein receptor-defective patients; mitogen activated T-blasts exhibited a markedly elevated DiI-HDL uptake compared to resting T-cells. Binding was saturable at 37 °C and of high affinity, with a Kd of 5 × 10−8 M. It was blocked by anti-apoAI polyclonal antibodies (F(ab)2 fraction), but not by anti-apolipoprotein (apo)E, anti-apoAII, or anti-apoB, and was inhibited competitively by HDL apoproteins and an apoAI-protein A fusion protein. T-cell associated DiI-HDL was increased by trypsin treatment (of the cells) and decreased by activation in the presence of HDL or low density lipoprotein. Comparison of the concentration dependencies of growth promotion and specific cell association of HDL indicated that two mechanisms of lipid exchange may be in operation: one a binding-dependent mechanism of cholesterol exchange, with maximal effect in the HDL concentration range (20-200 μg/ml) in which specific binding increases rapidly, and the other a binding-independent exchange of lipids effective at concentrations in which specific binding is saturated (300-5000 μg/ml).

Cholesterol flux between high density lipoproteins and cultured rat luteal cells.

Although high density lipoprotein (HDL) is a promoter of cholesterol efflux from peripheral cells, in steroidogenic tissues it has been shown to provide cholesterol for steroid synthesis. The present study examines the dynamics of cholesterol exchange between HDL particles and rat luteal cells. Cholesterol influx was measured by following the uptake of label from HDL particles labeled with [3H]cholesterol or [3H]cholesteryl linoleate. The efflux of cholesterol was simultaneously measured by incubating [3H]cholesterol-labeled luteal cells with unlabeled HDL. Conversion of endogenous and lipoprotein-derived steroids to progestins was also determined. The results showed that HDL promotes both influx and efflux of cholesterol in those cells. The amount of influx exceeds efflux, thus resulting in a net uptake of cholesterol from HDL by rat luteal cells. The relative utilization of endogenous vs. HDL-derived cholesterol for steroid synthesis was also examined. The results show that only a fraction of the HDL-derived cholesterol was converted directly to steroids. The fraction that was converted depended on the HDL concentration and had no apparent relation to the incubation time. These results show that although cholesterol flux in luteal cells is bidirectional in the presence of HDL, the influx exceeds the amount of efflux, and the internalized cholesterol is diluted with the endogenous cholesterol pool before it is converted to steroids.

Lipolytic surface remnants of triglyceride-rich lipoproteins are cytotoxic to macrophages but not in the presence of high density lipoprotein. A possible mechanism of atherogenesis?

Hypertriglyceridemic (HTG) serum, lipolyzed in vitro by purified bovine milk lipoprotein lipase, was found to be cytotoxic to cultured macrophages. Surviving macrophages contained numerous lipid inclusions similar to those found in foam cells. Individual lipoprotein fractions isolated from the lipolyzed HTG serum, including HDL, were also cytotoxic. Lipolysis of isolated lipoprotein fractions (either HTG or normal) allowed localization of cytotoxicity to postlipolysis remnant VLDL and chylomicron particles. The presence of a critical concentration of HDL in either the lipolysis mixture or the culture dishes inhibited the cytotoxicity. Below this critical concentration HDL itself became cytotoxic, producing lipid inclusions in surviving macrophages. The lipid fraction of the cytotoxic remnants contained the cytotoxic factor(s); neither FFA nor lysolecithin alone could account for this cytotoxicity. Postprandial lipemic sera from subjects with a brisk chylomicron response, when lipolyzed in vitro, were cytotoxic to cultured macrophages; neither fasted sera from these subjects, nor postprandial sera from normolipidemic subjects with a normal chylomicron response, were cytotoxic. Postheparin (in vivo lipolyzed) serum and its isolated lipoprotein fractions obtained 30 min after heparin injection in subjects with HTG were shown to be cytotoxic to macrophages; by 60 min most of the cytotoxicity had disappeared. The postprandial and postheparin observations support an in vivo significance for remnant-associated cytotoxicity. We hypothesize that cytotoxic remnants of lipolyzed VLDL and chylomicrons may be one of the major atherogenic lipoproteins. Further, we suggest that inhibition of the cytotoxicity of these remnants may be one important way that HDL prevents atherosclerosis.

Evidence for the presence of low density and very low density lipoproteins in human amniotic fluid

Previous analysis of amniotic fluid (AF) noted only the presence of high density lipoprotein (HDL). In this study AF lipoprotein profile was examined using gel filtration column chromatography and Ouchterlony gel diffusion. Unlike previous studies which showed only the presence of HDL, we found significant amounts of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). AF-LDL and AF-VLDL were identified by reactions with anti-h-apolipoprotein AI and AII antiserum and anti-h-apolipoprotein B-antiserum, respectively. Furthermore, bulk of the cholesterol mass was carried in VLDL (53.6 ± 7.7%) and LDL (32.5 ± 4.3%) with minor amounts (13.9 ± 1.3%) in HDL fraction. It is concluded that human AF contains all three lipoproteins with most of the cholesterol being carried in very low density lipoprotein fraction.

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37°C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d > 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d > 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d > 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4–24.0 nm) major components than for LDL with initially large (26.2–26.8 nm) major components. The facilitation factor in the plasma d > 1.20 g/ ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d > 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL /HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.

Cholesterol efflux from cultured skin fibroblasts in 12 patients clinically heterozygous for familial hypercholesterolemia

The [ 3H]cholesterol efflux from cultured skin fibroblasts was investigated in 12 patients, clinically heterozygous for familial hypercholesterolemia (FH). The [ 3H]cholesterol efflux from fibroblasts was studied in the presence of high-density lipoproteins, isolated either from control sera or from the patient's own serum. The [ 3H]cholesterol efflux from fibroblasts of 3 patients was increased; it was decreased for 4 patients and similar to that of control cells for 5 patients. This study indicates that in some patients with heterozygous FH, abnormalities of [ 3H]cholesterol efflux from fibroblasts can be detected. The heterogeneity of the results is consistent with the heterogeneity of the low-density lipoprotein receptor activity and with the variability of the clinical picture found in this disease.

Cholesterol synthesis in cultured human peripheral lymphocytes. Influence of LDL, HDL, cholesterol/phosphatidylcholine liposomes and complete serum

In lipoprotein-deficient milieu, freshly isolated human peripheral blood lymphocytes lose about 50% of their membrane cholesterol into the medium within 8 h. The cholesterol loss is counter-regulated by de novo synthesis commencing after a lag phase of 8–12 h, and reaching a steady state within 24 h at a diminished membrane cholesterol level. About 50 μg free cholesterol/ml, offered in the form of low-density lipoproteins (LDL) and cholesterol/phosphatidylcholine liposomes, suppressed cholesterol synthesis to about 20% of that in controls (lipoprotein-deficient culture). By contrast, pure phosphatidylcholine liposomes enhanced cholesterol synthesis to about 150% of control values. High-density lipoproteins (HDL) exerted a slightly suppressive effect on cholesterol synthesis only at high concentrations (greater than 100 μg HDL cholesterol/ml). HDL added to cultures containing fixed concentrations of LDL led to a dose-dependent neutralization of LDL suppression of cholesterol synthesis. Culture medium containing complete serum caused a suppression of cholesterol synthesis to about 50% of the control. The lesser reduction in cholesterol synthesis caused by complete serum compared with LDL or cholesterol/phosphatidylcholine liposomes can be explained by the presence of HDL in the former. Our results support the view that the cholesterol requirement of blood lymphocytes in their lipid-rich milieu is met by cholesterol neosynthesis as well as an exchange mechanism with surrounding lipoproteins. In our system, the cholesterol neosynthesis appears to be controlled by the ratio of LDL to HDL in the surrounding medium

Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membranes

The membrane properties of cholesterol auto-oxydation products, 7-ketocholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol and 25-hydroxycholesterol were examined. Monolayer studies show that these oxysterols are perpendicularly orientated at the interphase. Only 7β-hydroxycholesterol and 7α-hydroxycholesterol are tilted at low surface pressures. In mixed monolayers with dioleoylphosphatidylcholine, 7-ketocholesterol, 7β-hydroxycholesterol and 7α-hydroxycholesterol show a condensing effect in this order, although to a lesser extent that that observed for cholesterol. In liposomes these oxysterols also reduce glucose permeability and in the same order as their condensing effect. On the other hand 25-hydroxycholesterol shows no condensing effect in monomolecular layers whereas glucose permeability in liposomes is enormously increased. The permeability increase is already maximal at 2.5 mol% 25-hydroxycholesterol. Differential scanning calorimetry experiments reveal that all four oxysterols tested reduce the heat content of the gel → liquid-crystalline phase transition. It is concluded that 7-ketocholesterol, 7β-hydroxycholesterol and 7α-hydroxycholesterol have a cholesterol like effect, although less efficient than cholesterol, whereas 25-hydroxycholesterol showing no condensing effect acts as a spacer molecule. Packing defects in the hydrophobic core of the bilayer due to the presence of the C-25 hydroxyl group are believed to cause the permeability increase. The transfer of radiolabelled (oxy)sterols from the monolayer to lipoproteins or vesicles in the subphase was studied. The transfer rate increases in the following order 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 25-hydroxycholesterol. The difference in rate between 7-ketocholesterol and 25-hydroxycholesterol is 20-fold. A higher rate of transfer is observed in the presence of high density lipoproteins and small unilamellar vesicles. A transfer rate for cholesterol is hardly measurable under these conditions. The transfer measured is consistent with the involvement of a water-soluble intermediate.

Biotin and choline replace the growth requirement of Madin-Darby canine kidney cells for high-density lipoproteins.

MDCK cells maintained on extracellular matrix (ECM)-coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high-density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum-supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 micrograms/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 micrograms/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.

The secretion of apolipoprotein E by human monocytederived macrophages

The secretion of newly synthesized apolipoprotein E (apo E) by human monocytederived macrophages (HMD macrophages) was measured in the medium of cells which had been incubated for 24 h with or without either native or acetylated lowdensity lipoproteins (LDL or AcLDL, respectively), and subsequently with [ 35S ]methionine in the presence of high-density lipoproteins (HDL, 350 μg/ml) for 24 h, by isolating the lipoprotein fraction by centrifugation for 48 h at a density adjusted with KBr to 1.21 g/ml (d = 1.21). The d < 1.21 medium was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or precipitation with trichloroacetic acid (TCA). Fluorography of the gels demonstrated that the d < 1.21 fraction of the medium contained one major labeled band, which migrated with an apparent molecular weight of ~35,000. Immunoprecipitation of the d < 1.21 fraction showed that the labeled band was precipitated by anti-apo E, but not by anti-HDL. As the apo E band appeared to be the only labeled band in the d < 1.21 fraction, the amount of apo E secreted by the cells was quantitated by scintillation counting of the TCA-precipitable radioactivity in the d < 1.21 fraction as compared with that in the whole medium. The proportion of secreted apo E to the total secreted protein was similar whether the cells had been in culture for 3 or 16 days, but was increased if the cells had been incubated with LDL or AcLDL. The proportion of apo E of the secreted proteins was always more than 6% and was as much as 16% when the cells were preincubated with lipoproteins, suggesting that the increased cholesterol influx induced apo E secretion.

Influence of high-density lipoprotein on estradiol stimulation of luteal steroidogenesis.

The aim of this investigation was to determine whether luteal cells utilize cholesterol derived from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances luteal utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different doses of human HDL resulted in a dose-dependent increase in progesterone production. HDL in vitro enhanced the overall steroidogenic capacity. However, the percentage of increases in 17 alpha-hydroxyprogesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.

Effect of human high density lipoproteins, anti-apolipoproteins CII and CIII, and hydrolysis of very low density lipoprotein (VLDL) cholesterol ester on VLDL catabolism [formula omitted

Lipolysis of human very low density lipoproteins (VLDL) by lipoprotein lipase (LPL) was inhibited in the presence of high density lipoproteins (HDL), anti-apolipoprotein (apo) CII, and by increasing the VLDL free cholesterol content but not with anti-apo CIII or lipoprotein-free plasma. The experiments lend direct evidence that the composition of VLDL and their milieu are important determinants of lipolysis by LPL. Apo CIII may not be critical in LPL mediated VLDL catabolism.

Role of high density lipoproteins in regulation of hepatic fatty acid synthesis by chylomicron and very low density lipoprotein remnants.

High density lipoproteins (HDL) have been implicated in the transformation of native triglyceride-rich lipoproteins into their corresponding remnant particles during the action of peripheral lipoprotein lipase. The subsequent metabolism of these remnant particles by the liver results in the inhibition of hepatic lipogenesis. In the present study, remnant particles of chylomicrons or very low density lipoproteins (VLDL) have been generated in the perfused heart system both in the absence and presence of HDL. These have been characterized chemically, and the effects of both native lipoproteins and their respective remnants on fatty acid synthetic rates of hepatocytes have been assessed. Thirty to sixty-six per cent of the triglyceride moieties of native lymph chylomicrons or VLDL were hydrolyzed during a 45-min heart perfusion whether or not HDL was present in the perfusion media. Chylomicron remnants produced in the absence of HDL (25-300 micrograms/ml) caused only 10-20% inhibition of hepatic fatty acid synthesis, whereas remnants produced in the presence of HDL caused up to 78% inhibition at equivalent protein concentrations. The nonsuppressive remnants (produced in the absence of HDL) were converted to suppressive remnants upon incubation with HDL. Similar results were obtained with VLDL remnants produced in the absence and presence of HDL. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apoprotein profiles of the nonsuppressive and suppressive remnants indicated a marked loss of the C apoproteins during the conversion of native chylomicrons or the nonsuppressive remnants to the suppressive remnants. Thus, HDL seems to be required for the removal of apoprotein C during the transformation of triglyceride-rich lipoproteins to the suppressive remnants. There was, however, no enrichment of apo-E on the suppressive remnant particles. We, thus, could not verify the suggested role of HDL in enriching the suppressive remnants with apoproteins E.

Metabolism of high density lipoprotein by human fetal adrenal tissue.

The role of lipoproteins as a source of the cholesterol utilized for steroidogenesis by human fetal adrenal (HFA) tissue was investigated previously. It was found that low density lipoprotein (LDL) was the lipoprotein preferred as a source of cholesterol for steroidogenesis by the HFA. [125I]Iodo-LDL was taken up and degraded by HFA tissue in organ culture, and the degradation of [125I]iodo-LDL was stimulated when ACTH (1 microgram X ml-1) was present in the culture medium. Others have shown that high density lipoprotein (HDL) is utilized as a source of cholesterol for steroidogenesis by rat adrenocortical cells in vitro and by the adrenals of the adult rat in vivo. In the present investigation we evaluated the metabolism of [125I]iodo-HDL by HFA tissue. [125I]iodo-HDL uptake by the HFA tissue increased in a linear manner with time and as the concentration of [125I]iodo-HDL in the culture medium was increased. However, there was little degradation of [125I]iodo-HDL by HFA. Moreover, preincubation of HFA tissue in medium containing ACTH (1 microgram X ml-1) or HDL, in various concentrations, did not affect the rate of uptake and degradation of [125I]iodo-HDL. The rate of degradation of [125I]iodo-LDL was found to decrease to low levels as the concentration of nonradiolabeled LDL in the culture medium was increased, whereas nonradiolabeled HDL had little effect on the degradation of [125I]iodo-LDL. HFA tissue fragments were incubated in medium containing ACTH plus lipoprotein-poor serum (LPPS) alone or LPPS plus HDL in various concentrations (50-1000 microgram X ml-1). The medium was changed daily and assayed for dehydroisoandrosterone sulfate and cortisol. In the presence of HDL, steroid secretion rates were no greater than those attained by HFA maintained in medium containing LPPS. It is concluded that the HFA utilizes cholesterol derived from LDL for steroidogenesis and that HDL is not metabolized efficiently by the human fetal adrenal.

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. In vitro cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL 2 result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.

Lipid metabolism in cultured cells: Studies on lipoprotein-catalyzed reverse cholesterol transport in normal and homozygous familial hypercholesterolemic skin fibroblasts

Recent studies indicate that elevations in plasma low-density lipoproteins (LDL) increase the risk of coronary artery disease whereas high-density lipoproteins (HDL) confer protection. In vitro, lipoproteins stimulate reverse cholesterol transport (RCT) from cultured cells. In this paper, the relative contributions of isolated lipoproteins to the observed RCT activity of whole serum are compared in normal cells and in cells lacking a receptor for low density lipoproteins. Cultured transformed human lung fibroblasts (VA-4), prelabeled with [ 3H]cholesterol, were incubated with human HDL or LDL containing [ 14C]cholesterol. Rates of [ 3H]cholesterol efflux were stimulated 30-fold by HDL and 15-fold by LDL. Cholesterol efflux with HDL was approximately 3-fold greater than influx whereas with LDL influx and efflux were approximately equal. The intrinsic ability of both lipoprotein fractions to catalyze efflux was confirmed by extensive fractionation and purification on several chromatographic media. Homozygous familial hypercholesterolemic skin fibroblasts, which lack a high-affinity LDL receptor, and normal skin fibroblasts were labeled with [ 3H]cholesterol. Fibroblast monolayers incubated with serum gave linear release kinetics during a 6-h assay period. In the absence of serum, only minor amounts of cholesterol were released. In the presence of 10% whole serum, normal and mutant cells released cholesterol at essentially the same rates. Specific binding of 125I-labeled LDL was decreased 15-fold in mutant cells. Release of [ 3H]cholesterol to the medium, however, was stimulated to approximately the same extent by LDL in both cell types. Normal and mutant cells bound equivalent amounts of 125I-labeled HDL and the rates of cholesterol release in the presence of HDL were the same. These results show that both HDL and LDL contribute to the observed RCT activity of serum when efflux is measured by loss of radiolabeled cholesterol although only HDL stimulates significant net removal of the sterol. Since normal rates of cholesterol release were observed in familial hypercholesterolemic fibroblasts incubated with either serum or LDL, it is concluded that LDL-catalyzed cholesterol efflux does not require interaction with the high-affinity LDL receptor.

Effect of low density lipoprotein and high density lipoprotein on sodium dodecyl sulphate precipitation of very low density lipoprotein from human serum

In the presence of low density lipoprotein, the sodium sodecyl sulphate(SDS)-very low density lipoprotein(VLDL) complex sedimented, while in the presence of high density lipoprotein the complex floated. This SDS-VLDL aggregate floats at serum triglyceride to cholesterol ratio of 0.7-0.9 and sediments at a ratio of 0.2-0.5.