Lipid Microemulsions Research Articles

Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.

Murine macrophage RAW264 were investigated for their response to lipid-free apolipoproteins. Preincubation of the cells with 300 microM dibutyryl cyclic (dBc) AMP for 16 h induced specific binding of apolipoprotein (apo) A-I to the cells and apoA-I-mediated HDL formation with cellular lipids, neither of which was detected in the absence of dBcAMP. Dose-dependent changes of the apoA-I specific binding and the apoA-I-mediated cholesterol release were largely superimposable. ApoA-II also mediated lipid release after the treatment of the cells with dBcAMP and effectively displaced the apoA-I binding to the cells. In contrast, cellular cholesterol efflux to lipid microemulsion and to 2-(hydroxypropyl)-beta-cyclodextrin was uninfluenced by the dBcAMP treatment. To induce the cellular reactivity with apoA-I, the incubation with dBcAMP required at least 6 h. Actinomycin D, cycloheximide, puromycin, and brefeldin A suppressed both the induction of apoA-I-mediated lipid release and the apoA-I specific binding to the cells. Analysis of the expression level of ABC1 mRNA by using reverse transcription-polymerase chain reaction and oligonucleotide arrays revealed that ABC1 mRNA was already expressed in the dBcAMP-untreated cells, and the dBcAMP treatment for 16 h enhanced its expression 9-13-fold. We conclude that dBcAMP selectively induces apolipoprotein-mediated cellular lipid release and accordingly high-density lipoprotein generation by inducing specific binding of apolipoprotein, but does not influence diffusion-mediated lipid efflux. The cell-apolipoprotein interaction seems to depend on cellular protein biosynthesis and transport. A substantial increase in the level of ABC1 mRNA caused by the dBcAMP treatment indicates that ATP-binding cassette transporter 1, the protein product of ABC1, may directly be responsible for the interaction, but the question about the absence of the interaction with its baseline expression level remains.

The Binding of Oxidized Low Density Lipoprotein to Mouse CD36 Is Mediated in Part by Oxidized Phospholipids That Are Associated with Both the Lipid and Protein Moieties of the Lipoprotein

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

Spectrofluorometric quantification of neutral and polar lipids in zooplankton using Nile red

This study defines the conditions (wavelength selection and sensitivity) for spectrofluorometric quantification of neutral and polar lipids using Nile red (NR). The fluorescence intensity of the probe with standard lipids was examined in relation to lipid concentration and composition. Standards used included commercial triolein (TO) and phosphatidylcholine (PC), triacylglycerols (TG) and phospholipids (PL) from the krill Euphausia superba and Meganyctiphanes norvegica, and wax esters (WE) and PL from the copepods Paraeuchaeta antarctica and Drepanopus pectinatus. At any wavelength tested, polar and neutral lipids could be detected and fluorescence overlapped resulting in an interference of 10% to 20%. This interference between polar and neutral lipids was quantified for the standardisation. Wavelength selection for maximum emission with neutral and polar lipid classes achieved maximal sensitivity. Relationships between fluorescence intensity and lipid concentration were shown to be linear and specific to the lipid used. Large errors occurred when calibrations were performed with commercial lipids or with lipids obtained from other plankton species. Therefore, only the use of specific lipids as standards provided simultaneous and absolute quantification of neutral and polar lipids (relative errors were lower than 5%). The presence of PL in the sample (a ratio as low as 1:20) improved fluorescence of NR with TG by enhancing the solubility of the lipid microemulsion in water.

Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: implications with respect to macrophage recognition of apoptotic cells.

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4 degrees C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.

Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages.

Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.

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.

Activation of human plasma cholestryl ester transfer protein by human apolipoprotein A-IV

Function of apolipoprotein (apo) A-IV was studied for its role in cholesteryl ester transfer protein (CETP; lipid transfer protein, LTP) reaction between lipid microemulsions having the diameter of low density lipoprotein, being compared to apoA-I. CETP hardly catalyzed lipid transfer without apolipoproteins. ApoA-IV bound to the surface of the microemulsion in equilibrium with a similar affinity to that of other helical apolipoproteins, and activated the transfer reaction by CETP of cholesteryl ester, triacylgycerol and phosphatidylcholine between the emulsions. The rate of the transfer reaction of cholestery ester and triacylglycerol was directly proportional to the amount of the bound apoA-IV to the surface of the emulsion. For phosphatidylcholine, activation was less effective until 40% of total binding capacity of lipid emulsion was occupied by the apolipoprotein. Cholesteryl ester was highly preferred by CETP over triacylglycerol when equal amount of these lipids was present in the core of the apoA-IV-activated emulsion, resulting in almost no triacylglycerol transfer. However, when the emulsion has the core exclusively of triacylglycerol, triacylglycerol was transferrd by CETP with the rate in the same order as that of cholesteryl ester transfer. These findings were all comparable to the results with apoA-I, and also consistent with our previous observation for other amphiphilic helical apolipoproteins such as apoA-Ii, E and C-III.

Involvement of a cellular surface factor(s) in lipid-free apolipoprotein-mediated cellular cholesterol efflux

Involvement of cellular surface factors in cellular lipid efflux mediated by lipid-free apolipoprotein has been investigated. Lipid-free human apolipoprotein (apo) A-I generated net efflux of cholesterol and phospholipid from mouse peritoneal macrophages and rat aorta smooth muscle cells. Ratio of cholesterol to phospholipid was much lower in the lipid released by this mechanism from the smooth muscle cells than that from the macrophages, in agreement with our previous observation (Li, Q., Komaba, A. and Yokoyama, S. (1993) Biochemistry 32, 4597–4603). On the other hand, free apoA-I did not cause any lipid efflux from human erythrocytes. In contrast, apparent efflux of cellular cholesterol to HDL was similarly observed from all of these three cellular membranes. Trypsin treatment of the cultured macrophages completely inhibited apoA-I-mediated efflux of cholesterol and phospholipid. Smooth muscle cells also showed complete inhibition of the apoA-I-mediated cellular lipid efflux by trypsin treatment except that it required longer incubation with the enzyme. The same cellular treatment with trypsin even by prolonged incubation had only a limited effect on apparent cellular cholesterol efflux to HDL and apolipoprotein-free lipid microemulsions. Thus, free apolipoprotein-mediated cellular lipid efflux seems to depend on a trypsin-susceptible cellular surface factor(s) that erythrocytes may lack, being distinct from physicochemical cholesterol exchange reaction between cell and lipoprotein.

Independent Regulation of Cholesterol Incorporation into Free Apolipoprotein-mediated Cellular Lipid Efflux in Rat Vascular Smooth Muscle Cells

Cholesterol was poorly available to free apolipoprotein (apo)A-I-mediated cellular lipid efflux from cholesterol-loaded rat vascular smooth muscle cells generating cholesterol-poorer pre-beta-HDL particles than those generated from macrophages by the same reaction (Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). The factors known to induce transformation of the smooth muscle cells into a macrophage-like stage were used in order to modulate this reaction, such as human platelet-derived growth factor, macrophage colony-stimulating factor, and phorbol 12-myristate-13-acetate (PMA). When the cells were stimulated by PMA following the pretreatment with platelet-derived growth factor plus macrophage colony-stimulating factor, cholesterol efflux mediated by free apoA-I increased 3-fold without changing phospholipid efflux, resulting in generation of pre-beta-HDL particles more rich in cholesterol. This treatment had only a little or no effect on apparent cellular cholesterol efflux to HDL or lipid microemulsion, respectively. Overall cellular free cholesterol pool size was unaffected by the treatment, and probing by extracellular cholesterol oxidase did not detect gross change in the cellular surface cholesterol. This specific enrichment of cholesterol in the apoA-I-mediated cellular lipid efflux was reversed by protein kinase C inhibitors. Measurement of intracellular cholesterol esterification suggested that PMA induced translocation of intracellular cholesterol to a specific pool for apoA-I-mediated efflux, and a protein kinase C inhibitor reversed this effect.

Inverse targeting of drugs to reticuloendothelial system-rich organs by lipid microemulsion emulsified with poloxamer 338

Lipid microemulsions (LM) consisting of soybean oil and lecithin have been studied as a parenteral drug delivery system for site-specific delivery of non-water-soluble drugs. A major obstacle to targeting to non-RES organs or maintaining high concentrations of LM in vasculature is their rapid and extensive uptake by the RES in the liver and spleen. By replacing lecithin with hydrophilic poloxamer 338, it has been possible to avoid the normal deposition of LM in the liver and spleen (inverse targeting). Poloxamer 338-modified LM (PLM) containing ibuprofen octyl ester was intravenously administered to rats. Ibuprofen concentrations in the plasma and various organs were measured to elucidate the effect of inverse targeting to RES and targeting to other tissues in terms of the incorporated drug rather than the drug carrier. It was suggested that PLM can be exploited to direct lipophilic drugs in LM away from RES in the liver and spleen to other targeting tissues such as inflammatory tissues.

Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects.

This review highlights the state-of-the-art in pharmaceutical microemulsions with emphasis on self-emulsifying systems, from both a physical and biopharmaceutical perspective. Although these systems have several pharmaceutical applications, this review is primarily focused on their potential for oral drug delivery and intestinal absorption improvement. Physicochemical characteristics and formulation design based on drug solubility and membrane permeability are discussed. Case studies in which lipid microemulsions have successfully been used to improve drug solubilization/dissolution and/or intestinal absorption of poorly absorbed drugs/peptides are presented. Drug development issues such as commercial viability, mechanisms involved, range of applicability, safety, scale-up and manufacture are outlined, and future research and development efforts to address these issues are discussed.

Triglyceride transfer is required for net cholesteryl ester transfer between lipoproteins in plasma by lipid transfer protein. Evidence for a hetero-exchange transfer mechanism demonstrated by using novel monoclonal antibodies.

In order to investigate the role of lipid transfer protein (LTP) in plasma lipoprotein metabolism, monoclonal antibodies (mAbs) have been raised against LTP isolated from rabbit plasma. mAbs 2-8G and 3-9F inhibited both [3H]cholesteryl ester (CE) and [3H]triglyceride (TG) transfer from low density lipoprotein (LDL) to high density lipoprotein (HDL) mediated by LTP. Although 3-9F cross-reacted with human LTP, 2-8G was species-specific for rabbit LTP. mAb 14-8H inhibited only [3H]TG but not [3H]CE transfer and was cross-reactive with human LTP. mAbs 2-8G and 3-9F interfered with association of LTP with lipid microemulsions, again 2-8G with species specificity, whereas 14-8H did not affect LTP-microemulsion binding. Thus, mAbs 3-9F and 14-8H were used for further study in human plasma. By total inhibition of CE and TG transfer by 3-9F, LTP was shown to be responsible for net mass transfer of neutral lipids between lipoprotein classes in plasma, namely of CE from HDL to very low density lipoprotein (VLDL) and TG from VLDL to LDL and HDL. Selective inhibition of TG transfer by mAb 14-8H was also able to inhibit such net neutral lipid transfer. Such effect of these antibodies was demonstrated more remarkably in the presence of cholesterol esterification. Thus, TG transfer activity of LTP was shown to be required for net CE transfer, suggesting that net neutral lipid transfer in plasma between lipoproteins occurred mainly by a hetero-exchange mechanism. Inhibition of net neutral lipid transfer in plasma did not affect cholesterol esterification occurring predominantly on HDL. Consequently, mAb inhibition of TG transfer in plasma leads to CE accumulation in HDL. It is possible that hyperalphalipoproteinemia may be induced by a mutation in LTP that causes a selective defect in TG transfer activity.

Properties of human plasma lipid transfer protein in aqueous solution and at interfaces.

Human plasma lipid transfer protein (cholesteryl ester transfer protein) has been characterized for its solution and surface properties. The protein is monomeric in aqueous solution up to 0.62 g/L (11.7 microM) as demonstrated by sedimentation equilibrium. It binds to the surface of a lipid microemulsion having an average diameter of 26 nm made from triolein and egg yolk phosphatidylcholine, with an estimated dissociation constant 1.2 x 10(-8) M, and the maximum saturation binding level is 8 protein molecules per particle regardless of the presence of apolipoprotein A-I. Circular dichroism measurement indicated that the protein in solution is predominantly in the beta-sheet/beta-turn conformation with some alpha-helix, and this profile does not undergo drastic change by its binding to the lipid surface. The analysis of the behavior of the protein in its monomolecular layer at the air-buffer interface indicated that it is also monomeric at the interface. LTP molecules occupied the same area per amino acid as other apolipoproteins in the monolayer but had a higher collapse pressure of its monolayer (18 dyn/cm), and the protein stayed at the interface even after the overcompressing monolayer far beyond the collapsing pressure up to 40 dyn/cm.

Selective transfer of cholesteryl ester over triglyceride by human plasma lipid transfer protein between apolipoprotein-activated lipid microemulsions.

The substrate-specific rate of the human plasma lipid transfer protein (LTP) reaction was studied using pyrene-labeled substrate lipid analogues as probes for various lipids, by monitoring the ratio of the fluorescence intensities of their excimers to those of their monomers as an indicator of pyrene concentration in the microenvironment. Transfer of cholesteryl ester (CE) and triglyceride (TG) was demonstrated between human high-density lipoproteins, between low-density lipoproteins, and between these two lipoprotein, and the specific fractional transfer rate of CE was always higher than that of TG by a factor of 2.4-7.9. On the other hand, the transfer by LTP of CE, TG, and phosphatidylcholine (PC) was also demonstrated between lipid microemulsions having an average diameter of 25-26 nm using the same probes, but only when the emulsions were activated by apolipoproteins A-I, A-II, E, or C-III. The maximally activated rates of the transfer of CE and TG were the same when measured between the emulsions with cores composed exclusively of either lipid. The specific fractional transfer rate of pyrene-CE, however, was inversely proportional to the percentage of CE in the TG core of the emulsions, and the initial transfer of TG was almost completely inhibited by the presence of small percentages of CE in the TG core. Thus, the transfer of CE between the emulsions is highly selective over that of TG by orders of magnitude, much more selective than the reaction between any natural plasma lipoproteins, but this selectivity is not a rate-limiting step of the overall LTP reaction. The maximally activated LTP-catalyzed transfer rate of PC between the emulsions was somewhat higher than that of CE or TG and was not affected by the composition of the core lipids of the emulsion, TG or CE. When an excess amount of LTP was incubated with emulsion containing a small percentage of pyrene-CE in the TG core in the absence of the acceptor particles, excimer fluorescence rapidly decreased to the base line, and this change was suppressed when pyrene-CE was diluted with CE in the core. This result may indicate that LTP selectively disrupts pyrene-CE excimer formation on the basis of its selective interaction with the CE molecule over TG in the emulsion system as a putative background mechanism for the selective transfer of CE.

Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein.

A procedure for rapid isolation of lipid transfer protein (LTP) from commercially available rabbit plasma is described. Use of protease inhibitors was important for obtaining intact, stable LTP. After lipoproteins were precipitated from the plasma by dextran sulfate, column chromatographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M were employed. Overall purification from plasma was (3830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) = 74K and had an NH2-terminal amino acid sequence and amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP monoclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding assay, rabbit LTP was shown to predominantly associate with lipid microemulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glöckner analysis gave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alpha-helix, beta-sheet, beta-turn, and remainder content, respectively. Upon lipid binding, the helical content did not change drastically, although there was some disordering of beta structure.

Activation of human plasma lipid transfer protein by apolipoproteins.

Activation of human plasma lipid transfer protein (LTP) by apolipoproteins was studied. Pyrenelabeled cholesteryl ester was used as a probe substrate for the transfer reaction between lipid microemulsions, with a diameter of 26 nm, of triglyceride and phosphatidylcholine, and the reaction was monitored as a change in the ratio of the peaks of monomer and excimer in the fluorescence spectrum of pyrene. The transfer of pyrene-cholesteryl ester was hardly catalyzed by highly isolated LTP in the absence of apolipoprotein unless extreme overdose of LTP was given, regardless of the presence of bovine serum albumin. Human apolipoprotein (apo) A-I and apoA-II activated the LTP reaction in a dose-dependent manner. The activation was directly proportional to the titration of the surface of the substrate lipid emulsions by the apolipoproteins when the rate was plotted against the apolipoproteins bound to the surface. Human apoE also activated the LTP reaction in the same manner. The activation by human apoC-III was also proportional to the surface-bound protein, but the rate of the transfer was lower than those with other apolipoproteins. Displacement of apoA-I by apoC-III from the lipid emulsion surface, therefore, resulted in apparent deactivation of the LTP reaction. Thus, LTP requires apolipoproteins for its activation, and the activation seems proportional to the area of the surface of the lipid substrate particles modified by apolipoproteins. ApoA-I, -A-II, and -E are more potent activators than apoC-III for cholesteryl ester transfer.

Cholesterol is poorly available for free apolipoprotein-mediated cellular lipid efflux from smooth muscle cells

To study the mechanism for resistance of smooth muscle cells (SMC) to cholesterol efflux caused by lipid-free apolipoproteins [Komaba, A., et al. (1992) J. Biol. Chem. 267, 17560-17566], the efflux of phospholipids and cholesterol was induced from mouse peritoneal macrophages (MP) and rat aortic SMC by phospholipid/triglyceride microemulsion, by human plasma high- and low-density lipoproteins (HDLs and LDLs), and by lipid-free human apolipoprotein (apo) A-I. The efflux of both lipids by the lipid microemulsion showed essentially the same kinetic profile for these two types of cells except that the rate of phospholipid efflux was 5-6 times slower by weight than cholesterol in both cases. The same ratio of cholesterol to phospholipid was also found in the efflux to LDLs. Lipid-free apoA-I mediated cellular cholesterol efflux, but the rate was much slower from SMC than from MP. However, the rate of apoA-I-mediated phospholipid efflux was similar between these two cells generating HDL-like particles, resulting in a high phospholipid:cholesterol ratio, (4-5):1 by weight, in the lipid efflux from SMC, in contrast with (0.8-1):1 in the lipid efflux from MP. When standardized for the cellular free cholesterol, the Vmax of cholesterol efflux induced by lipid-free apoA-I was 10 times slower from SMC than from MP, but only by at most 2-fold slower when lipid microemulsion was the acceptor. Thus, free cholesterol of SMC is less available than that of MP for free apolipoprotein-mediated generation of HDLs with cellular lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of unesterified cholesterol on the compartmentation of a fluorescent cholesteryl ester in a lipoprotein-like lipid microemulsion.

The access of enzymes and lipid transfer proteins to neutral lipids located predominantly in the core compartment of lipoproteins may be determined to some degree by the solubility of the neutral lipids in the surface monolayer of phospholipid. This report concerns the hypothesis that unesterfied cholesterol can affect the partition of a cholesteryl ester between the surface monolayer of a lipid emulsion and the internal core compartment, thus controlling the degree to which the cholesteryl ester is presented at the emulsion surface. For microemulsions composed of dimyristoyl phosphatidylcholine and cholesteryl oleate, the addition of unesterified cholesterol results in an increase in the particle size from about 170 nm diameter to 210 nm diameter at 13.5 mol% unesterified cholesterol. Fluorescent quenching methods were devised to determine the apparent partition of a fluorescent cholesteryl ester (cholesteryl anthracene-9-carboxylate) between surface and core compartments. The addition of unesterified cholesterol resulted in the movement of the fluorescent cholesteryl ester from the surface monolayer to the core compartment. The apparent partition coefficient, defined as the ratio of the concentration of probe in the monolayer to that in the core, decreased from 1.03 in the absence of unesterfied cholesterol to 0.54 at 28 mol% unesterified cholesterol in the emulsion. In this process, the fluorescent cholesteryl ester becomes less accessible to a quencher (5-doxyl stearate) located in the surface monolayer. The decrease in the surface curvature resulting from incorporation of unesterified cholesterol into the particle does not influence this quenching process. We conclude that the presence of unesterified cholesterol in the emulsion causes the fluorescent cholesteryl ester to become less soluble in the surface monolayer.

Role of apolipoproteins in cholesterol efflux from macrophages to lipid microemulsion: proposal of a putative model for the pre-.beta.-high-density lipoprotein pathway

Lipid microemulsion of phospholipid and triglyceride with the size of low-density lipoprotein was capable of removing cholesterol from cholesterol-loaded mouse peritoneal macrophages, resulting in reduction of intracellularly accumulated cholesteryl ester. Apolipoproteins (apo) A-I, A-II, C-III, and E bound to the surface of the microemulsion did not modulate the interaction of the microemulsion with the cells in terms of the cholesterol efflux. The cholesterol removal by the microemulsion was enhanced by some 30% only when apoA-I, -A-II, and -E were present in excess to provide their free forms in the medium, but apoC-III did not show such an effect even by its excess amount. The kinetics including the results with apoC-III were consistent with a model that the apparent enhancement was due to generation of pre-beta high-density lipoprotein (HDL)-like particles upon the interaction of free apolipoproteins with macrophages [Hara, H., & Yokoyama, S. (1991) J. Biol. Chem. 266, 3080-3086]. However, pre beta-HDL-like particle was not detected after 6- and 24-h incubation in the medium where cholesterol efflux to the emulsion was maximally enhanced by the apolipoproteins, and cholesterol and phospholipids removed from the cells were all found with the microemulsions. It was also shown separately that the lipids in pre beta-HDL-like particles generated by apoA-I and macrophages were rapidly, within the order of minutes, transferred to the apo-lipoprotein-covered microemulsions when they were incubated together. Thus, the data were consistent with a model that the free form of certain apolipoproteins, such as apoA-I, -A-II, and -E but not apoC-III, generates pre beta-HDL-like particles with cellular lipids in situ and these particles act as mediators for cholesterol transfer from the cells to other lipoproteins.