Rates of cholesterol exchange between human erythrocytes and plasma lipoproteins

Rates of exchange of labelled cholesterol between human erythrocytes and three plasma lipoprotein species, LDL, HDL₂ and HDL₃, were measured over a range of lipoprotein concentrations and temperatures. The exchange rates reached limiting, concentration-independent values, which were the same for the three lipoproteins. The temperature dependencies correspond to activation energies of 12 kcal in the limiting rate region, and at lower lipoprotein concentrations to activation energies of 11 to 22 kcal. Calculations based on simple collision theory indicate that energetic barriers to the exchange are far smaller than indicated by these activation energies and that no particular orientation of lipoprotein molecules is required for the exchange. The occurrence of a limiting rate may be a result of the adsorption of lipoprotein molecules onto a limited number of sites on the cell surface, or of a slow process occurring in the membrane, possibly the diffusion of cholesterol. Present data do not permit a choice between these models.     

Fraction of cholesterol undergoing spontaneous exchange between small unilamellar phosphatidylcholine vesicles

The kinetics of the spontaneous exchange of [3H]cholesterol between small unilamellar vesicles of phosphatidylcholine has been reexamined. Although first-order exchange kinetics were observed (k = 0.0117 min-1), in good agreement with previous studies, about 20% of the total cholesterol was found to be nonexchangeable in the 8-h time frame of the experiments. The size of this nonexchangeable pool was found to depend on the type of phospholipid and the temperature. It seems probable that the two pools of cholesterol defined in these experiments reflect the complex phase structure of the cholesterol-phosphatidylcholine vesicles.     

Comparison of exchange of alpha-tocopherol and free cholesterol between rat plasma lipoproteins and erythrocytes.

The simultaneous exchange of (3h)tocopherol and (14C)cholesterol between rat plasma, rat plasma lipoproteins, and RBC was studied in vitro to compare quantitavely (a) the fractional exchange rates and (b) the half-times for isotope equilibration. In all incubations of RBC with plasma or with plasma lipoprotein fractions, (14C)cholesterol approached equilibrium more rapidly than (3H)tocopherol. When the RBC contained the initial radioactivity, the half-times for equilibration with plasma of cholesterol and of tocopherol were 1.0 and 2.2 hr, respectively. However, the fractional exchange rates (KRBC leads to plasma) were 0.097/hr for cholesterol and 0.188/hr for tocopherol, indicating that the RBC tocopherol pool is turning over almost twice as rapidly as the RBC cholesterol pool. The rat plasma lipoproteins were separated into five fractions by successive ultracentrifugation. Only two fractions, the high density lipoproteins (d 1.063-1.21) and the very low density lipoproteins (d is less than 1.006), participated to a significant extent in the exchange of either tocopherol or cholesterol with RBC. Cholesterol exchange between individual rat plasma lipoproteins and RBC had the same half-times for isotope equilibrium for the very low and high density lipoproteins, and the RBC fractional exchange rates were proportional to the amount of cholesterol in the lipoproteins. In tocopherol exchange between individual rat plasma lipoproteins and RBC, the very low density lipoprotein tocopherol did not equilibrate completely with the RBC. However, the initial rate of tocopherol exchange appeared to be the same for very low and high density lipoproteins. The very low density lipoproteins were disrupted by repeated freezing and thawing or by dehydrating and rehydrating, and analysis of the resulting lipoproteins indicated that free cholesterol was associated more closely than tocopherol with the phospholipid-protein portion of the molecule, which is thought to be on the surface. This difference in distribution of tocopherol and free cholesterol within very low density lipoproteins could account for their different rates of exchange and for the nonequilibrium of tocopherol between RBC and very low density lipoproteins.     

Cholesterol efflux to high-density lipoproteins and apolipoprotein A-I phosphatidylcholine complexes is inhibited by ethanol: role of apolipoprotein structure and cooperative interaction of phosphatidylcholine and cholesterol

There is a substantial body of evidence showing that moderate alcohol consumption is associated with a reduced risk of cardiovascular morbidity and mortality. One of the factors thought to contribute to this reduction in risk is an increase in the level of high-density lipoproteins (HDL) correlated with alcohol consumption. However, HDL levels are elevated in heavy drinkers, but their risk of vascular disease is greater compared with that of moderate drinkers. Ethanol at concentrations observed in heavy drinkers and alcoholics may directly act on HDL and apolipoproteins and in turn modify cholesterol efflux. In this paper, we show that ethanol significantly inhibited cholesterol efflux from fibroblasts to HDL and to apolipoprotein A-I (apoA-I) complexed with phosphatidylcholine (PC). Ethanol significantly inhibited binding of PC to apoA-I, inhibited incorporation of cholesterol only when apoA-I contained PC, and did not alter incorporation of cholesterol into HDL. ApoA-I structure was altered by ethanol as monitored by steady-state fluorescence polarization of tryptophan residues. The absence of ethanol effects on incorporation of cholesterol into HDL versus inhibition of cholesterol incorporation into the apoA-I-PC complex suggests that the effects of ethanol on cholesterol efflux mediated by HDL involve interaction with the cell surface and that efflux mediated by the apoA-I-PC complex is a combination of aqueous diffusion and contact with the cell surface. In addition, effects of ethanol on apoA-I suggest that pre-beta-HDL or lipid-free apoA-I may be more perturbed by ethanol than mature HDL, and such effects may be pathophysiological with respect to the process of reverse cholesterol transport in heavy drinkers and alcoholics.     

Influence of lecithin acyl chain composition on the kinetics of exchange between chylomicrons and high density lipoproteins

The kinetics of lecithin exchange between native lipoproteins was characterized for individual molecular species of lecithins of rat mesenteric lymph chylomicrons and rat plasma HDL. Studies were performed in the absence of lipid transfer proteins. Donor (chylomicrons) and acceptor (HDL) particles were present in ratios of 1:1 and 1:10 with respect to total phospholipid. Biphasic exchange kinetics were observed for all major lecithins common to chylomicrons and HDL at both proportions of donor to acceptor particles. During the early rapid phase of exchange, complete in about 30 min, 40-60% of the total lecithin pool was exchanged. Initial exchange rates were most rapid for the more hydrophilic species of the major lecithins normally present in both lipoproteins. Calculated activation energies correspondingly were least for a diunsaturated lecithin (18:1-20:4), intermediate for lecithins were 16:0 in position-1 (16:0-18:2 and 16:0-20:4), and highest for analogous lecithins with 18:0 in position-1. A 10-fold increase in the ratio of acceptor to donor particles affected neither the biphasic nature of the exchange nor the rates of exchange of individual molecular species (consistent with exchange by diffusion rather than by particle collisions). Total equilibration of individual molecular lecithin species was achieved by 24 hr (37 degrees C, donor to acceptor ratio of 1:1) with only a small change in the relative mass of lecithins in chylomicrons and HDL. Novel lecithins containing 18:3, incorporated into chylomicrons, were found to exchange exceedingly rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcohol and high-density lipoproteins.

High-density lipoproteins (HDL) have been shown to be negatively associated with coronary heart disease; some epidemiologic evidence also suggests that alcohol may protect against coronary heart disease, but other evidence shows the opposite. Alcohol ingestion and even alcoholism may be associated with higher serum HDL levels, but the levels tend to return to normal within 2 weeks with abstinence from alcohol. The relation between HDL and alcoholism, however, is complex, since in addition to alcohol itself several other factors have to be considered. Liver disease and cigarette smoking tend to decrease the serum HDL level in alcoholic persons, while certain hormonal and nutritional influences and the concomitant use of other microsomal-enzyme-inducing drugs may lead to increased HDL levels. On balance, while alcohol per se may increase the serum HDL level, alcoholism--particularly alcoholic liver disease--probably negates the HDL-related protection against coronary heart disease.     

Transfer of free and esterified cholesterol from low-density lipoproteins and high-density lipoproteins to human adipocytes

Cholesterol stored in human adipose tissue is derived from circulating lipoproteins. To delineate the cholesterol transport function of LDL and HDL, the movement of radiolabelled esterified cholesterol and free cholesterol from labelled LDL and HDL to human adipocytes was examined in the present study. LDL and HDL were enriched and labelled in esterified cholesterol with [14C]cholesterol by the action of plasma lipid transfer proteins and lecithin-cholesterol acyltransferase. Doubly labelled (3H,14C) LDL and HDL were prepared by exchanging free [3H]cholesterol into the 14C-labelled lipoproteins. 14C-labelled lipoprotein and 3H-labelled lipoprotein were also prepared separately and mixed to yield a mixed doubly labelled lipoprotein. Relative to the total amount added, proportionally more free than esterified cholesterol was transferred to the adipocytes upon incubation with any doubly labelled LDL and HDL. The calculated mass of free and esterified cholesterol transferred, however, varied with different labelled lipoproteins. 3H- and 14C-labelled LDL or HDL transferred 2-3-fold more esterified than free cholesterol while the reverse occurred with the mixed doubly labelled LDL or HDL. Thus, free cholesterol-depleted particles preferentially transferred cholesterol ester to the fat cells. In the presence of the homologous unlabelled native lipoprotein, the transfers of free and esterified cholesterol from labelled LDL or HDL were specifically inhibited. Selective transfer of esterified cholesterol relative to apoprotein was also observed when esterified cholesterol uptake from both LDL and HDL was assayed along with the binding of 125I-labelled lipoprotein. The cellular accumulation of cholesterol ether-labelled HDL (a non-hydrolyzable analogue of cholesterol ester) exceeded that of cholesterol ester consistent with significant hydrolysis of the latter physiological substrate. These results demonstrate preferential transfer of free cholesterol and esterified cholesterol over apoprotein for both LDL and HDL in human adipocytes. Furthermore, the data suggest that the cholesterol ester transport function of LDL and HDL can be enhanced by free cholesterol depletion and cholesterol ester enrichment of the particles, and affirms a role for adipose tissue in the metabolism of lipid-modified lipoproteins.     

Cholesterol transport between cells and high-density lipoproteins

Various types of studies in humans and animals suggest strongly that HDL is anti-atherogenic. The anti-atherogenic potential of HDL is thought to be due to its participation in reverse cholesterol transport, the process by which cholesterol is removed from non-hepatic cells and transported to the liver for elimination from the body. Extensive studies in cell culture systems have demonstrated that HDL is an important mediator of sterol transport between cells and the plasma compartment. The topic of this review is the mechanisms that account for sterol movement between HDL and cells. The most prominent and easily measured aspect of sterol movement between HDL and cells is the rapid bidirectional transfer of cholesterol between the lipoprotein and the plasma membrane. This movement occurs by unmediated diffusion, and in most situations its rate in each direction is limited by the rate of desorption of sterol molecules from the donor surface into the adjacent water phase. The net transfer of sterol mass out of cells occurs when there is either a relative enrichment of sterol within the plasma membrane or a depletion of sterol in HDL. Recent studies suggest that certain minor subfractions of HDL (with pre-beta mobility on agarose gel electrophoresis and containing apoprotein A-I but no apo A-II) are unusually efficient at promoting efflux of cell sterol. To what extent efflux to these HDL fractions is balanced by influx from the lipoprotein has not yet been established clearly. The prevention and reversal of atherosclerosis require the mobilization of cholesterol from internal (non-plasma membrane) cellular locations. To some extent, this may involve the retroendocytosis of HDL. However, most mobilization probably involves the transport of internal sterol to the plasma membrane, followed by desorption to extracellular HDL. Several laboratories are investigating the transport of sterol from intracellular locations to the plasma membrane. Studies on biosynthetic sterol (probably originating mostly in the smooth endoplasmic reticulum) suggest that there is rapid transport to the plasma membrane in lipid-rich vesicles. Important features of this transport are that it bypasses the Golgi apparatus and may be positively regulated by the specific binding of HDL to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

The exchange of unesterified cholesterol between human low-density lipoproteins and rat erythrocyte `ghosts''

1. The exchange of unesterified cholesterol molecules between rat erythrocyte ;ghosts' and human low-density lipoproteins has been studied under a number of different experimental conditions. 2. The process is pH-dependent, the rate being minimal at about pH5. 3. Cholesterol exchange does not vary greatly with temperature, the rate at 50 degrees being less than twice that at 2 degrees . 4. Large variations in the ionic strength or Ca(2+) concentration of the medium have little effect, but the exchange rate is greatly increased in the presence of a wide range of chemical compounds, e.g. urea, alcohols, acetone, dimethyl sulphoxide and tetra-alkyl-ammonium salts. 5. Acetone and dimethyl sulphoxide have a much greater effect at 37 degrees than at 8-10 degrees . 6. It is proposed that hydrophobic bonding is of great importance in maintaining the structure of ;ghosts' and lipoproteins. 7. The results are discussed in relation to current theories of membrane and lipoprotein structure.     

Disintegration of phosphatidylcholine liposomes in plasma as a result of interaction with high-density lipoproteins.

1. During in vitro incubation of liposomes or unilamellar vesicles prepared from egg-yolk or rat-liver phosphatidylcholine with human, monkey or rat plasma the phospholipid becomes associated with a high molecular weight protein-containing component. 2. The phosphatidylcholine . protein complex thus formed co-chromatographs with high-density lipoprotein on Ultrogel AcA34 and has the same immunoelectrophoretic properties as this lipoprotein. 3. Release of the phosphatidylcholine from liposomes was also observed when liposomes were incubated with pure monkey high-density lipoproteins. Under those conditions some transfer of protein from the lipoprotein to the liposomes was observed as well. 4. The observed release of phospholipid from the liposomes is a one-way process, as the specific radioactivity of liposome-associated phosphatidylcholine remained constant during incubation with plasma. 5. It is concluded that either the lipoprotein particle takes up additional phospholipid or that a new complex is formed from protein constituents of the lipoprotein and the liposomal phosphatidylcholine. 6. Massive release of entrapped 125I-labeled albumin from the liposome during incubation with plasma suggests that the observed release of phosphatidylcholine from the liposomes has a highly destructive influence on the liposomal structure. 7. Our results are discussed with special reference to the use of liposomes as intravenous carriers of drugs and enzymes.     

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.     

Imaging and manipulation of high-density lipoproteins.

The atomic force microscope (AFM) has been used to image a variety of biological systems, but has rarely been applied to soluble protein-lipid complexes. One of the primary physiological protein-lipid complexes is the high-density lipoproteins (HDL), responsible for the transport of cholesterol from the peripheral tissues and other lipoproteins to the liver. We have used the AFM to directly image discoidal reconstituted HDL (rHDL) particles for the first time. The height of these particles is consistent with a phospholipid bilayer structure, but careful high resolution measurements of particle diameters has indicated that they fuse when adsorbed to mica. Furthermore, it has been demonstrated that the AFM can be used to initiate this bilayer fusion in a controlled manner, allowing the fabrication of stabilized, nanometer scale, phospholipid bilayer "domains."     

Isolation and specificity of rat lecithin: cholesterol acyltransferase: comparison with the human enzyme using reassembled high-density lipoproteins containing ether analogs of phosphatidylcholine

Rat plasma lecithin: cholesterol acyltransferase, a 68 kDa glycoprotein, has been purified 14 000-fold by a modification of a procedure used for the human enzyme. The activity of lecithin: cholesteryl acyltransferase in human and rat plasma are the same, although activation of both enzymes by human apolipoprotein A-I is greater than that produced by rat apolipoprotein A-I. Using reassembled high-density lipoproteins composed of human apolipoprotein A-I, phosphatidylcholine ethers and a series of different phosphatidylcholines, the separate effects of molecular species specificity and microenvironment on the rate of cholesteryl ester formation was determined. Substitution of a fluid lipid, 1-palmityl-2-oleyl-sn-glycero-3-phosphorylcholine, for a solid lipid, 1,2-dipalmityl-sn-glycero-3-phosphorylcholine, produced an 8-fold increase in the activity of all molecular species of phosphatidylcholine. With either solid or fluid lipid environments, the activity decreased as a function of increasing chain length of saturated acyl groups. Addition of one or more double bonds greatly increased the activity of a given saturated homologue. One major difference between the molecular specificity of rat and human lecithin: cholesteryl acyltransferase was that the latter had a two-fold preference for phosphatidylcholines containing arachidonate at the sn-2-position.     

Cholesteryl ester and apolipoprotein E transfer between human high density lipoproteins and chylomicrons

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4 degrees C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1/10, a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2/1. From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.     

Evidence that lecithin:cholesterol acyltransferase acts on both high-density and low-density lipoproteins

In incubations of plasma containing lipoproteins at physiological concentrations it has been confirmed that high-density lipoproteins (HDL) are the major initial recipients of the esterified cholesterol formed in the reaction catalysed by lecithin:cholesterol acyltransferase. It has also been confirmed, however, that a small proportion of the esterified cholesterol of lecithin:cholesterol acyltransferase origin is incorporated directly into low-density lipoproteins (LDL), via a pathway that bypasses the HDL. This direct incorporation of esterified cholesterol into LDL is compatible with either of two general models. Model A proposes that lecithin:cholesterol acyltransferase does not interact directly with LDL but rather that it acts only on lipoproteins outside the LDL fraction. According to model A, while most of the esterified cholesterol so formed is incorporated into HDL, a small proportion is transferred directly to LDL. Model B, by contrast, proposes that a direct incorporation of esterified cholesterol into LDL is the result of a direct action of lecithin:cholesterol acyltransferase on the free cholesterol associated with LDL. To differentiate between these two models, experiments have been performed in which incubation mixtures containing LDL, HDL and a source of lecithin:cholesterol acyltransferase were supplemented with free [3H]cholesterol which had previously been incorporated into either LDL or HDL. It was found that, of the esterified [3H]cholesterol which was subsequently formed, the proportion recovered in the LDL fraction was much greater in the incubations to which the free [3H]cholesterol had been added as a component of LDL than in those to which it had been added as a component of HDL. This essentially excluded model A but was consistent with model B. It has been concluded that, while most of the lecithin:cholesterol acyltransferase may interact with particles in the HDL fraction, a small proportion of the enzyme interacts directly with LDL.