Phosphatidylcholine Complexes Research Articles

Temperature- and composition-dependent conformational transitions of amphipathic peptide–phospholipid nanodiscs

Nanodiscs are discoidal particles in which a lipid bilayer is encircled by amphipathic molecules such as proteins, peptides, or synthetic polymers. The apolipoprotein-A-I-derived peptide 18A is known to form nanodiscs in the presence of phospholipids, but the detailed mechanism of the formation and deformation of these nanodiscs in response to changes in the surrounding environment is not well understood. Here, we investigated the temperature- and composition-dependent structural changes of 18A–phosphatidylcholine complexes using fluorescence spectroscopy, dynamic light scattering, circular dichroism, static 31P NMR, and electron microscopy. We found that the nanodiscs in fast isotropic rotational motion increased in size above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayers, resulting in the formation of enlarged nanodiscs and a lamellar phase. The lamellar phase was found to be oriented along the magnetic field. Further increase in temperature induced the formation of lipid vesicles. These transformations were explained using a transition model based on the migration of the peptide from the rim of the nanodiscs to the liquid-crystalline bilayer phase. The study outcomes provide a basis for understanding the design principles of discoidal nanostructures for structural biology and nanomedicine applications.

Relative Quantification of Phosphatidylcholine sn-Isomers Using Positive Doubly Charged Lipid–Metal Ion Complexes

Phosphatidylcholines are the major phospholipid component of most eukaryotic cell membranes. Phosphatidylcholines have been shown to actively participate in regulatory and metabolic processes. Dysfunctional metabolic processes have been linked to human disease and can result in altered phosphatidylcholine structural features, such as permutation of fatty acid connectivity. Assignment and relative quantitation of structural isomers that arise from fatty acid permutation on the phosphatidylcholine backbone, so-called sn-isomers, is difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that phosphatidylcholines form abundant doubly charged metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign fatty acid moieties, relatively quantify sn-isomers in MS2 experiments, and mass spectrometrically separate phosphatidylcholines from other phospholipid classes in positive ion mode. Addition of Fe2+ salts (20 mol %) to ESI spray solutions affords highly abundant doubly charged metal ion phosphatidylcholine complexes (∼110% of protonated compounds) and allows sensitive fragment ion detection (limit of detection = 100 pM). Higher energy collisional dissociation, collision-induced dissociation, and ultraviolet photodissociation of doubly charged complexes yield two fragment ions for every fatty acid moiety. The latter two tandem MS methods preferentially yield sn-2 associated product ions enabling relative sn-isomer quantification. The analytical utility of doubly charged phosphatidylcholine-metal ion complexes is demonstrated for polar lipid extracts, including extracts from diabetes type 1 and type 2 mouse models, and sn-isomer abundances are derived.

Huperzine A–phospholipid complex-loaded biodegradable thermosensitive polymer gel for controlled drug release

The huperzine A–phospholipid complex loaded biodegradable thermosensitive PLGA–PEG–PLGA polymer gel was studied as injectable implant system for controlled release of huperzine-A (HA). First, HA molecules were successfully incorporated into the soybean phosphatidylcholine (SP) molecules to form the huperzine-A–soybean phosphatidylcholine complexes (HA–SPC), which was proved by FT-IR, DSC, XRD, solubility study, TEM, etc. The results indicated that hydrogen bonds and electrostatic interaction between HA and SP molecules play an important role in the formation of HA–SPC. Secondly, the HA–SPC was loaded into biodegradable PLGA–PEG–PLGA thermosensitive gel as injectable implant material to control the release of HA. The in vitro and in vivo drug release behaviors of the prepared products were studied. The in vitro release studies demonstrated that the HA–SPC-loaded gel significantly reduced the initial burst of drug release and extended the release period to about 2 weeks. The in vivo pharmacokinetics study of HA–SPC-loaded gel in rabbits showed that plasma concentration of HA (2.54–0.15ng/mL) was detected for nearly 2 weeks from delivery systems upon single subcutaneous injection. What's more, the in vitro release pattern correlated well with the in vivo pharmacokinetics profile. The present study indicates that HA–SPC loaded PLGA–PEG–PLGA thermal gel may be an attractive candidate vehicle for controlled HA release.

The phosphatidylcholine synthase of Pseudomonas putida A ATCC 12633 is responsible for the synthesis of phosphatidylcholine, which acts as a temporary reservoir for Al3+

In Pseudomonas putida A ATCC 12633 cells grown with tetradecyltrimethylammonium bromide and exposed to Al(3)Cl, phosphatidylcholine (PC) levels increased, which alleviated stress caused by the Al(3+). Here we cloned and sequenced a gene from this strain that encodes a phosphatidylcholine synthase (PCS) and characterized a pcs-deficient mutant. In the pcs-deficient mutant, PC could not be detected, whereas the mutant could be successfully complemented and expressed the enzyme, indicating that PC synthesis occurs exclusively via the PCS pathway in this organism. Although under non-stressing growth conditions the pcs-deficient mutant showed growth like that of the wild-type strain, the mutant was much more sensitive when challenged with Al(3+), which strongly supports the supposition that PC is involved in the response of P. putida to Al(3+) and acts as a temporary reservoir of available ions through the formation of Al(3+) : PC complexes.

Slow diuron release formulations based on clay–phosphatidylcholine complexes

Slow release formulations of the herbicide diuron were designed to reduce the risk of water pollution resulting from conventional formulations. The new formulations were based on herbicide incorporation in phosphatidylcholine (PC) vesicles that were sorbed on the clay mineral montmorillonite. The active ingredient contents of the formulations reached up to 28% w:w. Thermal analysis revealed that the closely packed arrangement of PC on the clay enhanced diuron sorption by disrupting the interactions among PC molecules. Adsorption experiments using diuron were performed in soils with different physico-chemical properties to evaluate the relationship between the sorption and the mobility of the herbicide. In soil column experiments with sandy soil, the herbicide in PC–clay-based formulations mainly accumulated in the top soil, and there was a one-third reduction in leaching compared to the commercial formulation. The differences in leaching (compared to the commercial formulation) were lower when using loam soil due to its higher sorption of the herbicide and hysteresis effects. In the sandy soil, bioassay experiments showed a higher herbicidal activity in the top-soil layer for the PC–clay formulations than for the commercial formulation. Therefore, PC–clay formulations of this herbicide can be used at lower doses than recommended, thereby reducing the environmental risks associated with the application of diuron formulations.

Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds

Apolipoprotein AI Milano (ApoAI(Milano) ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI(Milano) per kilogram of seed have been identified in a line selected for commercialization. The ApoAI(Milano) fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-ApoAI(Milano) product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI(Milano) readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAI(Milano) :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI(Milano) can be produced using a plant-based expression system.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Composition, structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein–phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type approximately R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I(M) had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I(M). The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.

Dose-Related Effects of Repeated ETC-216 (Recombinant Apolipoprotein A-IMilano/1-Palmitoyl-2-Oleoyl Phosphatidylcholine Complexes) Administrations on Rabbit Lipid-Rich Soft Plaques: In Vivo Assessment by Intravascular Ultrasound and Magnetic Resonance Imaging

This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose. These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Abstract 1047: Infusion of Reconstituted High Density Lipoproteins Into Humans Enhances the In Vitro Anti-Inflammatory Capacity of the High Density Lipoprotein Fraction.

Background: Intravenous infusions of reconstituted high density lipoproteins (rHDL) have been shown to promote regression of coronary atheroma. Here we investigate the effects of rHDL infusion on the anti-inflammatory capacity of the isolated HDL fraction. Methods: Eight fasting Type 2 Diabetics (mean age 52 ± 2 years) received IV saline or apolipoprotein A-I (apoA-I)/phosphatidylcholine complexes (rHDL) at an apoA-I dose of 80mg/kg, on separate occasions in random order. The HDL fraction was isolated from subjects at baseline, 4 &amp; 72hrs after infusion and assayed for chemical composition. The anti-inflammatory capacity of HDL preparations was assessed by incubation for 16hrs with human coronary artery endothelial cells. Cell expression of vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule (ICAM-1) 5 hrs after TNF-α stimulation was quantitated by flow cytometry. Results : Saline infusion had no effect on concentration or anti-inflammatory capacity of isolated HDL. rHDL infusion increased the concentration of HDL apoA-I (at baseline, 4h &amp; 72h, [apoA-I] = 966 ± 105, 2562 ± 330 &amp; 1701 ± 180 (ug/mL) respectively), as well as HDL cholesterol and HDL phospholipids. The addition of equivalent volumes of HDL (100uL) isolated at each time point inhibited VCAM-1 (Panel A) and ICAM-1 (Panel B) expression in proportion to the apoA-I concentration of the isolated HDL (p&lt;0.01 ANOVA across time points for both VCAM-1/ICAM-1 expression and apoA-I concentration) (see figure ). Conclusions: Infusion of rHDL in humans increases the concentration of HDL apoA-I. The anti-inflammatory capacity of isolated HDL is enhanced in proportion to its apoA-I concentration.

Interactions between phosphatidylcholines and cholesterol in monolayers at the air/water interface

Mixtures of cholesterol and synthetic phospholipids, differing in saturation of phosphatidylcholine (PC) acyl chains, such as distearoyl phosphatidylcholine (DSPC), stearoyl–oleoyl phosphatidylcholine (SOPC) and dioleoyl phosphatidylcholine (DOPC) have been studied as floating Langmuir monolayers at the air/water interface. In order to examine the influence of a polar group, distearoyl phosphatidylethanolamine (DSPE) was chosen. The films were spread at room temperature on aqueous subphases and characterized by the surface pressure–area ( π– A) isotherms and compression modulus ( C s − 1 ) values. The interactions were examined by analyzing the mean molecular areas and quantified by the excess free energy of mixing values. The obtained results indicate that the affinity of cholesterol to saturated/unsaturated phosphatidylcholines does not differ significantly, and revealed strong influence of the kind of a polar group on the cholesterol–phospholipid interactions. On the other hand, the apolar group structure was found to modify the stoichiometry of sterol–PC complexes.

Interactions of amphotericin B with saturated and unsaturated phosphatidylcholines at the air/water interface

Three synthetic phospholipids, differing in the degree of saturation of phosphatidylcholine (PC) acyl chains, namely: dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC) and palmitoyl-oleoyl phosphatidyl choline (POPC) were mixed with the antimycotic polyene antibiotic, amphotericin B (AmB), and investigated as monolayers at the air/water interface. The mixed films were spread on water (pH 6) at room temperature, and the surface pressure–area (π/A) isotherms were recorded upon compression. The interactions were examined by analysing the phase transitions of the monolayers and calculating the films compressibility. The obtained results indicate the existence of stronger interaction between AmB and the saturated phospholipid as compared with unsaturated ones, and reveal the influence of apolar structure of PC chains on the stoichiometry of AmB–PC complexes.

Fetal therapy in the correction of liver metabolic function in ischemic patients

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein–phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.

DNA–phospholipid recognition: modulation by metal ion and lipid nature. Complexes structure and stability calculated by molecular mechanics

The structures and formation energies of nucleic acid–phospholipid complexes both in the absence and in the presence of Mg 2+ ions were calculated taking double-stranded trinucleoside diphosphates NpNpN or heptanucleotides ApAp(NpNpN)pApA, composed of 64 possible combinations of genetic code, and phosphatidylcholine (PC) and sphingomyelin (SM) as model compounds. The dependence of intramolecular interactions on the primary structure of nucleic acid molecules and on the presence of a cationic bridge was revealed. The formation energies and structure of oligonucleotides were found by molecular mechanics calculations with the AMBER force field. The structures of phospholipid and MgCl 2 molecules were calculated by the semiempirical PM3 method, while the energies of phospholipid–oligonucleotide complexes were calculated by the molecular mechanics method. Calculations of complexes were carried out with consideration of solvation effects. Considerable gain in the formation energy of triple complexes is achieved due to the presence of the electroneutral metal bridge. A tendency toward increasing the stability of “triple” PC complexes (but not SM ones), containing guanosine- and cytidine-enriched triplets was revealed. Depending on the structure of NpNpN trinucleotides, the formation energy values of NpNpN–MgCl 2–PC and ApAp(NpNpN)pApA–MgCl 2–PC complexes differ by 1.7–2.6 kcal mol −1, which can be considered as the atomic-scale manifestation of the recognition phenomenon. Presence of metal (II) ion bridge results in a greater stabilization of the phospholipid–nucleic acid complexes for SM in comparison to PC (the total energy difference equals to 4–16 kcal mol −1). Depending on the structure of NpNpN trinucleotides, the formation energies of NpNpN–MgCl 2–SM and ApAp(NpNpN)pApA–MgCl 2–SM complexes differ by 1.7–2.1 kcal·mol −1, which is essential at physiological conditions and can also be considered as the recognition effect.

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136–150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa aminoterminal fragments (residues 1–191) of apoE3 and apoE4 bound to the LDLR with ~100-fold greater affinity than the 22-kDa fragment of apoE2. The pKa values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pKa of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic α-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity. The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pKa of K146 significantly, but it increased the pKa of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.—Lund-Katz, S., S. Wehrli, M. Zaiou, Y. Newhouse, K. H. Weisgraber, and M. C. Phillips. Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE.

Thermal Lipid Order−Disorder Transitions in Complexes of Various Disulfide Tethered Macrocyclic Diacylglycerol Analogues and Dipalmitoyl Phosphatidyl Choline. Role of Diacylglycerol Chain Motions

The role of diacylglycerol (DAG) chain motions in binary mixtures of DAG with dipalmitoylphosphatidyl choline (DPPC) has been studied using differential scanning calorimetry (DSC). The DAGs used in this study were synthesized to have different extents of chain restriction. This was accomplished via tethering of two long hydrocarbon chains by a reducible disulfide bond at different depths in the chain. The disulfide tether provides the advantage of comparing the effect of incorporation of each of these chain restricted DAGs in DPPC vesicles with their reduced analogue, in which the S-S linkage could be opened up to -SH by in situ treatment with a reducing agent, dithiothreitol (DTT). DSC analysis with such mixtures reveals that restriction of chains at different depths in the DAGs lead to very different properties of the overall DPPC/DAG coaggregate. DAGs in which the disulfide tether is located at the middle of the chain have only partial chain restriction. Incorporation of this type of DAG in DPPC destabilizes the resulting vesicles while the end-tethered DAGs upon incorporation in DPPC vesicles lead to stabilization of the coaggregates. Similar studies with the reduced, open-chain analogues reveal that release in chain restriction leads to extra stabilization in the coaggregates.

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.

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.