Rate Of Cholesterol Exchange Research Articles

The Interpretation of Cholesterol Balance Derived Synthesis Data and Surrogate Noncholesterol Plasma Markers for Cholesterol Synthesis under Lipid Lowering Therapies

The cholesterol balance procedure allows the calculation of cholesterol synthesis based on the assumption that loss of endogenous cholesterol via fecal excretion and bile acid synthesis is compensated by de novo synthesis. Under ezetimibe therapy hepatic cholesterol is diminished which can be compensated by hepatic de novo synthesis and hepatic extraction of plasma cholesterol. The plasma lathosterol concentration corrected for total cholesterol concentration (R_Lath) as a marker of de novo cholesterol synthesis is increased during ezetimibe treatment but unchanged under treatment with ezetimibe and simvastatin. Cholesterol balance derived synthesis data increase during both therapies. We hypothesize the following. (1) The cholesterol balance data must be applied to the hepatobiliary cholesterol pool. (2) The calculated cholesterol synthesis value is the sum of hepatic de novo synthesis and the net plasma—liver cholesterol exchange rate. (3) The reduced rate of biliary cholesterol absorption is the major trigger for the regulation of hepatic cholesterol metabolism under ezetimibe treatment. Supportive experimental and literature data are presented that describe changes of cholesterol fluxes under ezetimibe, statin, and combined treatments in omnivores and vegans, link plasma R_Lath to liver function, and define hepatic de novo synthesis as target for regulation of synthesis. An ezetimibe dependent direct hepatic drug effect cannot be excluded.

Interaction of cholesterol with sphingomyelin in bilayer membranes: evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles

Cholesterol undergoes exchange between membranes containing sphingomyelin at a much slower rate than between membranes lacking sphingomyelin. To investigate the role of the hydroxy group at the 3-position of sphingomyelin in the interaction between sphingomyelin and cholesterol, we have measured the rates of [4-14C]cholesterol exchange between unilamellar vesicles prepared with N-stearoylsphingomyelin or with synthetic analogues in which the hydroxy group is replaced with an O-alkyl group or with hydrogen. Vesicles prepared from 3-deoxy- and 3-O-methyl-N-stearoylsphingomyelin had the same rate of [14C]-cholesterol desorption. The half-times for exchange from vesicles prepared with 3-O-methyl- and 3-deoxy-N-stearoylsphingomyelins and 10 mol % of cholesterol were only slightly faster (a factor of only 1.5) than that found from vesicles prepared from N-stearoylsphingomyelin and 10 mol % cholesterol. The rate of cholesterol desorption from vesicles could be accelerated by preparing vesicles from bulky 3-O-alkyl analogues of sphingomyelin. Vesicles containing 3-O-ethyl-N-stearoylsphingomyelin and 3-O-tetrahydropyranyl egg sphingomyelin gave rate enhancements of approximately 14 and 35, compared with the rates observed in vesicles made from N-stearoyl- and egg sphingomyelin, respectively. These data indicate that insertion of sterically bulky groups at the 3-position of sphingomyelin (such as ethoxy and tetrahydropyranyloxy) in place of hydroxy interferes markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes; however, the hydroxy group of sphingomyelin is not critical for the strong interaction of cholesterol with sphingomyelin.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipids containing nitrogen- and sulfur-linked chains: kinetics of cholesterol exchange between vesicles

We have examined the kinetics of [ 14C]cholesterol exchange between unilamellar vesicles formed from the following synthetic glycerophosphatidylcholines: (a) those having acyl (OC(O)R), acylamino (NHC(O)R), carbamoyl (NHC(O)OR), and acylthio (SC(O)R) chains at the sn-2 position, and (b) those having alkyl (OR) and thioalkyl (SR) chains at the sn-1 position. Replacement of the glycerol oxygen atom at the sn-2 position of PC with a NH group did not affect the rate of cholesterol exchange to a significant extent, suggesting that the amide group of sphingomyelin is not primarily responsible for the very slow rate of exchange of cholesterol observed from sphingomyelin vesicles. Replacement of the glycerol oxygen at the sn-2 position of phosphatidylcholine with a sulfur atom caused the rate of spontaneous cholesterol exchange to increase by a factor of 1.6. Substitution of an O-alkyl chain for the acyl chain at the sn-1 position of 2-acylthiophosphatidylcholine or substitution of a thioalkyl chain for the O-alkyl sn-1 chain of 1-alkyl-2-acylaminodeoxyphosphatidylcholine also did not result in a marked difference in cholesterol exchange rate. The data suggest that interactions other than intermolecular hydrogen bonding are involved in determining the rates of intermembrane cholesterol exchange. Significantly, these kinetic studies also lend support to the continued use in model membranes of synthetic sulfur- and nitrogen-substituted phosphatidylcholines, which have been employed to study properties of lipolytic enzymes, since synthetic acylamino- and acylthio-phospholipids form vesicles that give cholesterol exchange rates that closely resemble those found in vesicles prepared with diester-phosphatidylcholines.

Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells

The membrane lipid composition and [3H]cholesterol exchange rate were studied in both normal human erythrocytes and those infected with the human malaria Plasmodium falciparum. The host cell membrane was separated from parasite membranes using the Affigel (731) bead method. The purity of the membrane preparation was very high, as judged by SDS-PAGE, and in several instances was estimated to be greater than 98% as determined by the activity of the parasite membrane-specific enzyme, choline phosphotransferase. No difference was found in the content of phosphatidylethanolamine and only small changes were observed for phosphatidylcholine and phosphatidylserine. The sphingomyelin content in red cell membranes of both trophozoite- and schizont-infected cells was up to 47% less than that of uninfected cells, and the cholesterol/phospholipid ratio was decreased 55%. Trophozoite- and schizont-infected cells exchanged 29 and 33% less cholesterol, respectively, than uninfected cells. These changes in lipid composition and cholesterol exchange could have a marked effect on the function of the red cell membrane of malaria-infected cells and may be responsible, in part, for the increased fluidity and permeability of P. falciparum-infected erythrocytes.

Movement of cholesterol between vesicles prepared with different phospholipids or sizes.

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.

Molecular species composition of membrane phosphatidylcholine influences the rate of cholesterol efflux from human erythrocytes and vesicles of erythrocyte lipid

The efflux of [ 3H]cholesterol from prelabelled human erythrocytes having modified phosphatidylcholine compositions was measured during 24-h incubations in the presence of unlabelled acceptor liposomes composed of equimolar amounts of egg phosphatidylcholine and cholesterol. The cells were modified by replacement of part of the native phosphatidylcholine with either dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine or dilinoleoylphosphatidylcholine catalyzed by phosphatidylcholine-specific transfer protein from bovine liver. The results indicated that the efflux of [ 3H]cholesterol was faster from erythrocytes in which the dipalmitoylphosphatidylcholine content was increased from 7 to 25% of the total, than from cells enriched in palmitoyloleoylphosphatidylcholine or dioleoylphosphatidylcholine. Incorporation of dilinoleoylphosphatidylcholine to a level of 13% of the total phosphatidylcholine slowed the rate of efflux of [ 3H]sterol. The phosphatidylcholine replacements produced no significant differences in cholesterol/ phospholipid ratio before or after 24 h of incubation with the acceptor egg phosphatidylcholine-cholesterol vesicles. Using vesicles prepared from erythrocyte lipid, modified to reflect the changes in the phosphatidylcholine composition induced in the whole cells, the same influence of composition on the rate of cholesterol exchange was evident. Enhancement of the dipalmitoylphosphatidylcholine content from 7 to 25% of the total phosphatidylcholine pool increased the rate of [ 3H]cholesterol efflux, while the addition of the same amount of dilinoleoylphosphatidylcholine slowed it compared to controls. The magnitude of the effect was comparable in intact cells and erythrocyte lipid vesicles enriched in dipalmitoylphosphatidylcholine, while the influence of dilinoleoylphosphatidylcholine was more marked in the intact cells. These results demonstrate that changes in the molecular species composition of the phosphatidylcholine pool can influence the rate of exchange of cholesterol but not necessarily the cellular content of sterol in the human erythrocyte. The influence of this phospholipid appears to be expressed independently of the presence of membrane protein or an underlying cytoskeleton.

Decreases in rates of lipid exchange between Mycoplasma gallisepticum cells and unilamellar vesicles by incorporation of sphingomyelin.

The kinetics of exchange of radiolabeled cholesterol and phospholipids between Mycoplasma gallisepticum cells and an excess of small unilamellar phospholipid/cholesterol vesicles were studied using cells enriched with different phospholipid classes but the same fatty acyl content. The rates of the rapidly exchanging cholesterol and phospholipid pools were markedly slower in sphingomyelin-containing cells than in phosphatidylcholine-containing cells. The decreased rates of spontaneous cholesterol and protein-mediated phospholipid exchange from the outer leaflet of the mycoplasma membrane are attributed to stronger interactions between sphingomyelin and cholesterol than between phosphatidylcholine and cholesterol. On the other hand, an increase in cholesterol exchange rate was found on incorporation of a cyclopentano-1,3-diacyl-2-phosphatidylglycerol, which interacts less with cholesterol than does the analogous glycero-1,2-diacyl-sn-3-phosphatidylglycerol.

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.

Symmetrical distribution and rapid transbilayer movement of cholesterol in Mycoplasma gallisepticum membranes

The exchange of cholesterol between [ 14C]cholesterol-labeled Mycoplasma gallisepticum cells and an excess of sonicated egg phosphatidylcholine/cholesterol vesicles (molar ratio of 0.9) was measured. More than 90% of the radioactive cholesterol underwent transfer from intact cells to the vesicles. The kinetics of the transfer was biphasic. About 50% of the radioactive cholesterol was exchanged with a half-time of about 4 h. The residual was exchanged at a slower rate with a half-time of about 9 h at 37°C. Bovine serum albumin had a pronounced effect in enhancing both the fast and slow rates of cholesterol exchange, but did not affect the pool sizes significantly. The half-time for equilibration of the two pools in the presence of 2% albumin, calculated using a reversible two-pool method of analysis, was 6.2 h. The effect of albumin was also obtained with isolated membrane preparations and with cells treated with growth inhibitors, suggesting that this effect is independent of albumin preservation of cell viability. The rate enhancement of albumin was concentration dependent with maximal effects observed with ⩾2%, where the rates of exchange of both the rapidly and slowly exchanging pools were twice as fast. The mechanism by which albumin may affect the exchange rates is discussed.

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, HDL 2 and HDL 3, 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.

Cholesterol exchange between fat cells, chylomicrons and plasma lipoproteins

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14C-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants > very low density lipoprotein > low density lipoprotein > high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.

Cholesterol metabolism in the macrophage. II. Alteration of subcellular exchangeable cholesterol compartments and exchange in other cell types.

Macrophage membrane cholesterol is present in two subcellular cholesterol pools, a rapidly exchanging compartment comprising about two-thirds of the total cholesterol, and a slowly exchanging compartment comprising one-third of the total. The morphological identification of the kinetically distinguishable pools proceeded by alteration of each compartment. Trypsin treatment markedly decreased the rate of cholesterol exchange without removing cholesterol from the membrane. Recovery of normal exchange rates took more than 7 hr and required protein synthesis. This suggested that a plasma membrane receptor is involved in positioning of lipoproteins for exchange, and is consistent with the plasma membrane localization of the rapidly exchanging compartment. Extensive pinocytosis by nondegradable dextran, dextran sulfate, or sucrose resulted in the accumulation of many secondary lysosomes, thus increasing the relative proportion of intracellular membranes. The measurable granule membrane area, cholesterol content, phospholipid content, and the relative size of the slowly exchanging cholesterol compartment all increased. The amount of intracellular membrane altered by extensive phagocytosis of latex particles also increased the size of the slowly exchanging cholesterol compartment. This suggested that the slowly exchanging pool of cholesterol represented the intracellular membranes primarily of lysosomal origin. Rabbit alveolar macrophages and thioglycollate-stimulated peritoneal macrophages contain many secondary lysosomes as a result of multiple bouts of in vivo phagocytosis and pinocytosis. In both of these cells the fast and slow pools are equal in size. The increased cholesterol content was attributable to the increase in the relative size of the slowly exchanging compartment. L-cells and melanoma cells also exchange their cholesterol with that of serum lipoproteins. Both cells contain few cholesterol-rich intracellular membranes, and had lower cellular cholesterol contents. In these cells the slowly exchanging pool was a minor contribution to cell cholesterol. Studies with these cells provided further evidence for the lysosomal membrane and plasma membrane localization of the slowly and rapidly exchanging cholesterol compartments.