Effects of charged lipids on the interaction of cholesteryl ester transfer protein with lipid microemulsions.

This study reports the effects of charged lipids on the transfer of cholesterlyl-1 pyrene decanoate (Py-CE) between apolipoprotein-free microemulsion particles mediated by cholesteryl ester transfer protein (CETP). The surface charge characteristics of microemulsion particles composed of cholesteryl oleate and egg yolk phosphatidylcholine were altered by incorporating phosphatidyl-serine, oleate or stearylamine into the phosphatidylcholine that forms the surface monolayer of the particle. The transfer of Py-CE was measured continuously by following the decrease in excimer fluorescence that accompanies the transfer of the probe from donor to acceptor particles [Rajaram, Chan and Sawyer (1994) Biochem. J. 304, 423-430]. The inclusion of 20 mol% phosphatidylserine relative to the phospholipid in the surface monolayer of the emulsion caused a 64% decrease in the first-order rate constant describing the transfer. An increase in ionic strength caused a partial reversal of this effects, indicating that electrostatic factors are only partially responsible for the interaction with lipid. Complete inhibition of transfer was observed when 10 mol% sodium oleate was incorporated into the surface monolayer. The incorporation of stearylamine into the emulsion caused a 32% increase in the transfer rate. The binding of CETP to the different emulsion surfaces was also examined using a surface plasmon resonance biosensor. The presence of negatively charged lipid (phosphatidylserine or oleic acid) decreased the rate of association of CETP with the emulsion without a significant change in the dissociation rate constant. The presence of the positively charged lipid stearylamine increased the rate of association of CETP with the lipid surface. It is concluded that a negative surface charge on the monolayer decreases the rate of transfer by decreasing the affinity of CETP for these particles.

Characterisation of lipid-protein interactions using a surface plasmon resonance biosensor.

The binding of proteins to lipid surfaces is of fundamental importance when considering the mechanisms of lipases and lipid transfer proteins. Current procedures for determining the binding constants suffer from problems related to poor sensitivity, poor reproducibility and to the use of restrictive assumptions in the analysis. We demonstrate here the use of the surface plasmon resonance biosensor to determine the association and dissociation kinetics of binding of cholesteryl ester transfer protein to the surface of emulsion particles of defined size and composition. Uncertainty about the emulsion valency, that is, the number of sites on the lipid particle occupied by the immobilised transfer protein on the biosensor surface, means that the association kinetics can only be characterised in terms of the product of the association rate constant and the concentration of sites, whereas the dissociation rate constant can be obtained unambiguously. The technique will prove useful in characterising the binding of proteins to lipid surfaces.

Effect of unesterified cholesterol on the activity of cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from high-density lipoprotein to triacylglycerol-rich lipoproteins and the transfer of triacylglycerols in the reverse direction. The activity of CETP has been studied using a continuous fluorescence assay which measures the excimer fluorescence of cholesteryl 1-pyrene decanoate in a synthetic donor microemulsion as the indicator of cholesteryl ester transfer. Emulsions were composed of cholesteryl oleate and egg phosphatidylcholine and had an average particle size of 14 +/- 1 nm as calculated from the molar volume of the components. The effect of changing the physical state of the emulsion surface was examined by including unesterified cholesterol in the donor and acceptor particles. The rate of CETP-induced transfer of the fluorescent cholesteryl ester between microemulsion particles increased when unesterified cholesterol was present at concentrations up to 17 mol% relative to phospholipid. The presence of cholesterol also changed the exchange kinetics from an apparent single-exponential to a double-exponential phenomenon. Binding of CETP to the emulsion surface was accompanied by an enhancement of fluorescence which was used to measure the binding equilibria. The enhancement of exchange due to the presence of cholesterol did not correlate with any increased binding of CETP to the emulsion surface. The presence of unesterified cholesterol in the donor did not affect the rate of transfer of the fluorescent cholesteryl ester when unlabelled emulsion was replaced by high-density lipoprotein as the acceptor. The studies demonstrate the use of microemulsions of defined size and composition for the study of the mechanism of action of CETP.

Relationship between the size and phospholipid content of low-density lipoproteins.

In studies performed in vivo and in vitro, it has been found that the Stokes' diameter of human low-density lipoproteins (LDL) correlates positively and significantly with the molar ratio of phospholipid/apo B in LDL but not with the LDL molar ratios of either cholesterol/apo B or triacylglycerol/apo B. It has been concluded that the phospholipid content of LDL is an important determinant of LDL size.

Sodium oleate dissociates the heteroexchange of cholesteryl esters and triacylglycerol between HDL and triacylglycerol-rich lipoproteins.

Mixtures of human high-density lipoproteins (HDL) and triacylglycerol-rich lipoproteins (TGRL) have been incubated in the presence of partially pure cholesteryl ester transfer protein (CETP). There were net mass transfers of cholesteryl ester from HDL to TGRL and of triacylglycerol from TGRL to HDL which were accompanied by the formation of minor subpopulations of small HDL particles. When the mixture of HDL, TGRL and CETP was supplemented with fatty acid-poor bovine serum albumin (40 mg/ml) there was a 7% reduction in the transfer of cholesteryl esters out of HDL (P less than 0.05) and a 14% increase in the transfer of triacylglycerol into HDL (P less than 0.05); there was also a reduction in the formation of very small HDL particles. In contrast, when the mixture of HDL, TGRL and CETP was supplemented with 0.16 mM sodium oleate the transfer of cholesteryl esters out of HDL was increased by 31% (P less than 0.001) and the transfer of triacylglycerol into HDL was decreased by 25% (P less than 0.01); under these conditions the formation of very small HDL particles was enhanced. It has been concluded that in the presence of sodium oleate, there is a dissociation of the CETP-mediated heteroexchange of cholesteryl esters and triacylglycerol between HDL and TGRL.

Sodium oleate promotes a redistribution of cholesteryl esters from high to low density lipoproteins.

Cholesteryl esters readily exchanges between the low density lipoproteins (LDL) and high density lipoproteins (HDL) in human plasma in a process of equilibration catalysed by the cholesteryl ester transfer protein (CETP). In the present studies, in which mixtures of human LDL and HDL have been incubated in vitro with partially pure CETP, it has been found that Na oleate disrupts the CETP-mediated equilibrium between LDL and HDL and promotes a concentration dependent redistribution of cholesteryl esters from HDL to LDL. The end result of the redistribution is the appearance of a cholesteryl ester enriched LDL fraction and an HDL fraction which is protein-rich, lipid-depleted and markedly reduced in particle size.

The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high-density lipoproteins.

Purified human cholesteryl ester transfer protein (CETP) has been found, under certain conditions, to promote changes to the particle size distribution of high-density lipoproteins (HDL) which are comparable to those attributed to a putative HDL conversion factor. When preparations of either the conversion factor or CETP are incubated with HDL3 in the presence of very-low-density lipoproteins (VLDL) or low-density lipoproteins (LDL), the HDL3 are converted to very small particles. The possibility that the conversion factor may be identical to CETP was supported by two observations: (1) CETP was found to be the main protein constituent of preparations of the conversion factor and (2) an antibody to CETP not only abolished the cholesteryl ester transfer activity of the conversion factor preparations but also inhibited changes to HDL particle size. In additional studies, the changes to HDL particle size promoted by purified CETP were inhibited by the presence of fatty-acid-free bovine serum albumin; by contrast, albumin had no effect on the cholesteryl ester transfer activity of the CETP. The possibility that albumin may inhibit changes to HDL particle size by removing unesterified fatty acids from either the lipoproteins or CETP was tested by adding exogenous unesterified fatty acids to the incubations. In incubations of HDL with either VLDL or LDL, sodium oleate had no effect on HDL particle size. However, when CETP was also present in the incubation mixtures the capacity of CETP to reduce the particle size of HDL was greatly enhanced by the addition of sodium oleate. It is concluded that the changes in HDL particle size which were previously attributed to an HDL conversion factor can be explained in terms of the interacting effects of CETP and unesterified fatty acids.

Isolation of a high-density-lipoprotein conversion factor from human plasma. A possible role of apolipoprotein A-IV as its activator.

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.

Enzymes involved in plasma cholesterol transport.

Regulation of plasma cholesterol transport is to a large extent a function of factors that regulate plasma cholesterol esterification and the transfers of cholesteryl esters between plasma lipoprotein fractions. Plasma cholesterol esterification is catalysed by the action of lecithin: cholesterol acyltransferase on lipids on the surface of HDL, while the transfers of cholesteryl esters require activity of a specific lipid transfer protein. Esterification of the cholesterol on the surface of HDL generates a concentration gradient down which unesterified cholesterol moves from tissues into the plasma. Once within the plasma and esterified, the newly formed cholesteryl esters are incorporated initially into the core of HDL particles before being redistributed to other classes of lipoproteins. The end result of these processes of esterification and transfer is that most of the cholesterol in human plasma is accommodated within the core of LDL, where its transport is a function of the highly regulated uptake by tissues of intact LDL particles. The capacity of HDL to act as substrates for lecithin: cholesterol acyltransferase varies inversely with HDL particle size. Thus, factors such as the concentration of triglyceride-rich lipoproteins and activities of the lipid transfer protein, hepatic lipase, lipoprotein lipase and the HDL conversion protein, which are known to influence HDL particle size, may also be important as regulators of plasma cholesterol esterification.

Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins.

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.

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.

Influence of lipoprotein concentration on the exchanges of triacylglycerol between rabbit plasma low density and high density lipoproteins.

Molecular exchanges of triacylglycerol between rabbit serum low density lipoproteins (LDL) and high density lipoproteins (HDL) have been studied in 37 degrees C incubations performed in the presence of rabbit lipoprotein-free serum as a source of the triacylglycerol transfer protein. The molar rate of exchange of triacylglycerol between the two fractions increased with increasing incubation concentrations of LDL but was decreased as the HDL concentration was increased. When the concentration of both LDL and HDL was increased in parallel there was an increase in the molar rate of triacylglycerol exchange between the two fractions which flattened at higher concentrations, suggesting that the process was saturable. Fractionation of rabbit lipoprotein-free serum on a column of Sephadex G-200 resulted in the elution of the triacylglycerol transfer activity in a single peak. Addition of LDL to the lipoprotein-free serum had no effect on the position of elution of the triacylglycerol transfer activity. Addition of HDL, however, resulted in an elution of the transfer activity as two peaks, one in the original position and the other in the same position as HDL. The results of the kinetic studies have been interpreted in terms of a binding of the triacylglycerol transfer protein to HDL, but not to LDL.

Partial purification and characterization of a triacylglycerol-transfer protein from rabbit serum.

Rabbit lipoprotein-free serum was found to contain a protein fraction that mediates the transfer of [3H]triacylglycerol from low-density lipoprotein to high-density lipoprotein. Fractionation of rabbit lipoprotein-free serum by DEAE-Sephadex chromatography, ammonium sulphate precipitation, concanavalin-A-Sepharose chromatography, Sephadex G-200 gel filtration, phenyl-Sepharose chromatography and Sephadex G-100 gel filtration, yielded a preparation that had a 500-fold increase in transfer activity compared to that of the starting sample. The transfer activity appeared to reside in a glycoprotein of molecular weight in the range 100 000-155 000 and an isoelectric point at pH 9.

Differences in the metabolism of very-low-density lipoproteins by isolated beating-heart cells and the isolated perfused rat heart. Evidence for collagenase-released extracellular lipoprotein lipase.

1. The metabolism of VLD lipoproteins (very-low-density lipoproteins) was studied in intact isolated beating-heart cells and isolated perfused rat heart from starved animals by using [14C]triacylglycerol fatty acid-labelled VLD lipoprotein prepared from rats previously injected with [1-14C]palmitate. 2. 14C-labelled VLD lipoprotein was metabolized by the isolated perfused heart, but was only minimally metabolized by the heart cells unless an exogenous source of lipoprotein lipase was added. 3. Measurements of lipoprotein lipase at pH 7.4 with the natural substrate 14C-labelled VLD lipoprotein indicated that during collagenase perfusion of the heart the enzyme was released into the perfusate, the activity released being proportional to the concentration of collagenase used. Lipoprotein lipase activity in homogenates of hearts that had been perfused with collagenase showed a corresponding loss of activity. 4. At high perfusate concentrations of collagenase, inactivation of the released lipoprotein lipase occurred. 5. Lipoprotein lipase activity was largely undetectable in the homogenate of the isolated heart cells. 6. It is concluded that the lipoprotein lipase responsible for the hydrolysis of VLD lipoprotein triacylglycerol is predominantly located externally to the heart muscle cells and that its release can be facilitated by perfusion of the heart with bacterial collagenase.

Species differences in the activity of a serum triglyceride transferring factor.

Low density lipoproteins (LDL), endogenously labelled with 3H in the triglyceride moiety, were isolated from rabbit serum and subsequently incubated in vitro at 37 degrees C with unlabelled preparations of rabbit high density lipoproteins (HDL) or very low density lipoproteins (VLDL). In incubations performed in the presence of phosphate buffer, there was no significant transfer of [3H]triglyceride from LDL to either HDL or VLDL; but when rabbit lipoprotein-free serum (the dialysed 1.21 g/ml infranatant) was added, transfer was apparent to both HDL and VLDL. The triglyceride transferring activity of the lipoprotein-free serum was abolished by heating at 85 degrees C for 10 min; all the transferring activity was found in the fraction which precipitated with ammonium sulphate at a concentration of less than 50% saturation. In direct contrast to the rabbit studies, rat serum failed to show a comparable process of triglyceride transfer. In subsequent experiments, mixtures of labelled LDL and unlabelled VLDL isolated either from rabbits or from rats were incubated with lipoprotein-free rabbit, rat or human serum. The lipoprotein-free serum of both the rabbit and man was effective in promoting transfer of 30--50% of LDL [3H]triglyceride into VLDL, regardless of the species origin of the lipoproteins. By contrast the lipoprotein-free serum of rats was only slightly more effective than buffer alone in promoting such transfers. It has been concluded that rabbit and human serum contains a triglyceride transferring factor of far greater activity than that in rat serum.

Effect of protein deficiency on absorption, transport and distribution of alpha-tocopherol in the rat.

The absorption, transport and distribution of alpha-[3H]tocopherol were greatly decreased in protein deficiency. This was reflected in the subcellular distribution of alpha-[3H]tocopherol in livers of protein-deficient rats. The ratio, bound:free for alpha-[3H]tocopherol, also decreased in both serum and liver cytosol. After protein refeeding, absorption, transport and distribution patterns of alpha-[3H]tocopherol for the protein-deficient rats were restored to patterns similar to those of control animals.

Involvement of binding lipoproteins in the absorption and transport of alpha-tocopherol in the rat.

1. Specific lipoproteins binding alpha-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered alpha-[(3)H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of alpha-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of alpha-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of alpha-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.