Characterization and synthesis of mono- and diphytanyl ethers of glycerol

The methanolyzed lipids of the extreme halophile, Halobacterium cutirubrum, were separated into glycerol diether and glycerol monoether fractions. The diether was shown by synthesis to be 2,3-di-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl)-sn-glycerol. The monoether fraction was separated by thin-layer chromatography on boric acid-impregnated silicic acid into about equal amounts of alpha- and -isomers. The alpha-isomer was found to be identical with the synthetic 3-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl)-sn-glycerol, and the -isomer was identical with the synthetic 2-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl) glycerol.     

Can intact liposomes be absorbed in the gut?

Inulin, EDTA, or maltose were given intragastrically to mice, either free or entrapped in mono-shell liposomes of egg phosphatidylcholine or the non-digestible diether and dialkyl analog of phosphatidylcholine. Results indicate that liposomes may protect a drug from premature digestion but cannot carry it through the intestinal wall.     

Effect of phosphatidylcholine on fatty acid and cholesterol absorption from mixed micellar solutions.

Using the experimental model of the everted sac prepared from rat jejuna, kinetic studies on [14C]oleic acid uptake from bile salt micelles were conducted in the presence and absence of phosphatidylcholine. The concentration of oleic acid was varied between 0.625 and 5 mM. At every level of fatty acid concentration studied the addition of 2 mM phosphatidylcholine produced a significant inhibition of fatty acid uptake. It was further noted that the intact phospholipid molecule was required for this effect as lysophosphatidylcholine produced little, if any, inhibition of [14C]oleic acid uptake. The effect of varying the concentration of phosphatidylcholine on fatty acid uptake was also studied. The degree of inhibition was noted to be correlated grossly with media concentrations of this phospholipid although the decrease of fatty acid uptake was not strictly proportional to concentration of this material in the medium. Studies were also performed analyzing in vitro absorption of [14C]oleic acid and [3H]cholesterol simultaneously from mixed micelles composed of sodium taurocholate, oleic acid, monoolein and cholesterol. Control medium contained no phospholipid while experimental medium contained either diester or diether phosphatidylcholine, 2 mM. Both types of phosphatidylcholine caused significant inhibition of fatty acid and cholesterol uptake. In vivo absorption studies were also performed using the isolated jejunal segment technique. A mixed micellar solution containing [3H]cholesterol and [14C]oleic acid was used as the test dose. Phospholipid in the test dose for controls was supplied as lysophosphatidylcholine and for experimentals it was in the form of diether phosphatidylcholine. Significantly less radioactively labeled cholesterol and fatty acid was absorbed by experimentals as compared to controls over a 10-min period. It is concluded that the intact molecule of phosphatidylcholine inhibits intestinal uptake of cholesterol and fatty acid from mixed micellar solutions under both in vitro and in vivo conditions.     

A range of ether core lipids from the methanogenic archaebacterium Methanosarcina barkeri

The core ether lipids of the methanogenic archaebacterium Methanosarcina barkeri were found to consist of C₂₀,C₂₀ glycerol diether and trace amounts of C₂₀,C₂₅ glycerol diether, C₂₀ glycerol monoether, C₄₀,C₄₀ diglycerol tetraethers with 1–3 cyclopentane rings, and a novel C₂₀,C₂₀ tetritol diether.     

Protein-mediated lipid transfer. The effects of lipid-phase transition and of charged lipids.

The protein-mediated phospholipid exchange between small unilamellar vesicles was investigated by fluorescence polarization measurements with diphenylhexatriene as optical probe. Thermotropic phase-transition measurements were taken after mixing two vesicle preparations of distinct and different phase-transition temperatures or having different states of charge. From the heights of each phase-transition step, we were able to follow the lipid-exchange process in the presence, as well as in the absence (natural exchange), of so-called transfer protein isolated from beef liver. A strong enhancement of the lipid transfer was observed at the corresponding lipid-phase-transition temperature, which is explained by the presence of fluctuating fluid and ordered domains co-existing at the lipid-phase-transition temperature. A unidirectional lipid transfer of the neutral component was observed between negatively charged phosphatidic acid and neutral phosphatidylcholine vesicles. Fluorescence polarization measurements showed the disappearance of the phosphatidylcholine phase transition, whereas the phosphatidic acid phase transition broadened and its phase transition temperature became lower.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids. Permeability and 13C-nuclear magnetic resonance measurements

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.     

Physical studies on phosphonium phosphatidylcholine. A unique [31P]phosphorus nuclear-magnetic-resonance probe for model and biological membranes.

1. Distearoyl phosphatidylcholine and the phosphonium analogue, in which the nitrogen atom is replaced by phosphorus, show similar gel-liquid crystalline transition temperatures as detected by differential scanning calorimetry. 2. The temperature-dependence of the 31P n.m.r. (nuclear-magnetic-resonance) linewidths of the phosphate resonances of sonicated vesicles of distearoyl phosphatidylcholine and the phosphonium analogue are similar. Below the phase-transition temperature the linewidths decrease as the temperature is raised. Above the phase-transition temperature the phosphate resonances are relatively temperature-independent. The phosphonium 31P n.m.r. signal exhibits the same pattern of temperature-dependence. 3. The 31P n.m.r. phosphonium resonance is sensitive to the paramagnetic shift reagent, K3Fe(CN)6. Use of K3Fe(CN)6, together with Nd(NO3)3, enabled the determination of the trans-bilayer distribution of egg-yolk phosphatidylcholine and its phosphonium analogue in co-sonicated vesicles. Both are distributed comparably across the bilayer of the vesicles. 4. The phosphonium 31P n.m.r. signal is much sharper than the corresponding phosphate resonance in both sonicated and unsonicated dispersions of the phosphatidylcholine analogue. 5. The properties of the phosphonium analogue of phosphatidylcholine are discussed in terms of its suitability as a probe of membrane structure.     

Antagonism between high pressure and anesthetics in the thermal phase-transition of dipalmitoyl phosphatidylcholine bilayer

The antagonizing action of hydrostatic pressure against anesthesia is well known. The present study was undertaken to quantitate the effects of hydrostatic pressure and anesthetics upon the phase-transition temperature of dipalmitoyl phosphatidylcholine vesicles. The drugs used to anesthetize the phospholipid vesicles included an inhalation anesthetic, halothane, a dissociable local anesthetic, lidocaine and an undissociable local anesthetic, benzyl alcohol. All anesthetics decreased the phase-transition temperature dose-dependently. In the case of lidocaine, the depression was pH dependent and only uncharged molecules were effective. The application of hydrostatic pressure increased the phase-transition temperature both in the presence and the absence of anesthetics. The temperature-pressure relationship was linear over the entire pressure range studied up to 340 bars. Through the use of Clapeyron-Clausius equation, the volume change accompanying the phase-transition of the membrane was calculated to be 27.0 cm3/mol. Although the anesthetics decreased the phase-transition temperature, the molar volume change accompanying the phase-transition was not altered. The anesthetics displaced the temperature-pressure lines parallel to each other. The mole fraction of the anesthetics in the liquid crystalline membrane, calculated from the van't Hoff equation, was independent of pressure. This implies that pressure does not displace the anesthetics from the liquid membrane, and the partition of these agents remains constant. The volume change of the anesthetized phospholipid membranes is entirely dependent upon the phase-transition and not on the space occupied by the anesthetics.     

Long-living liposomes as potential drug carriers

Neutral, unilamellar liposomes (vesicles) composed of a dialkyl analog of phosphatidylcholine and cholesterol, and containing ¹⁴C-maltose as entrapped marker, were administered intravenously to mice. After one and two days, radioactivity in blood and liver remained 3–4 times higher than after administration of liposomes of egg (diester) phosphatidylcholine and cholesterol. It appears that the vesicles were taken into liver cells by endocytosis, and that phospholipases are involved in the capture as well as in the breakdown of conventional liposomes. Liposomes that are semi-resistant to catabolic enzymes may become useful in the manipulation of drug delivery.     

Effect of progesterone on DPPC membrane: evidence for lateral phase separation and inverse action in lipid dynamics

Interactions of progesterone with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques namely Fourier transform infrared spectroscopy, turbidity at 440 nm, and differential scanning calorimetry. The results reveal that progesterone changes the physical properties of DPPC bilayers by decreasing the main phase-transition temperature, abolishing the pre-transition, broadening the phase-transition profile, disordering the system both in gel and liquid crystalline phase, increasing the dynamics at low concentrations whereas stabilizing the membrane at high concentrations, and inducing phase separation. Progesterone does not change the hydration of the CO groups, while it strengthens the hydrogen bonding between the PO2- groups of lipids and the water molecules around.     

Acyl chain and headgroup specificity of human plasma lecithin:cholesterol acyltransferase. Separation of matrix and molecular specificities

To determine how substrate fluidity and molecular structure independently regulate cholesteryl ester formation, the substrate specificity of lecithin:cholesterol acyltransferase with respect to a number of model reassembled high density lipoproteins (R-HDLs) is reported. The R-HDLs are composed of 1 mol % apolipoprotein A-I, 89 mol % of sphingomyelin or a nonhydrolyzable diether analog of phosphatidylcholine (PC) plus 10 mol % of test lipids that are potential acyl donors; a trace of [3H]cholesterol, which permits quantification of cholesteryl ester formation is also included. With respect to the lipid class of the acyl donor, the rate of ester formation decreases in the order phosphatidylethanolamine greater than phosphatidylcholine greater than N,N,-dimethylphosphatidylethanolamine greater than phosphatidylglycerol - phosphatidic acid greater than phosphatidylserine greater than dipalmitin greater than tripalmitin. Within an R-HDL composed of 90% PC ether or sphingomyelin, the relative rates of ester formation are greatest for dipalmitoyl and dimyristoyl PC, with distearoyl PC being almost unreactive; in a solid lipid environment, the rate with respect to unsaturation of the PC is greatest for oleate. In a fluid lipid environment, all unsaturated PCs were utilized nearly equally. All lipids tested were most reactive within an R-HDL composed of an unsaturated PC ether and least reactive within an R-HDL composed mostly of sphingomyelin. These results suggest that the rates of ester formation by lecithin:cholesterol acyltransferase are separate functions of the identity and the microscopic environment of the acyl donor. This is the first example of the use of diether analogs for the separation of the effects of macromolecular and molecular structure on the specificity of lecithin:cholesterol acyltransferase.     

Quantitative conversion of diether or tetraether phospholipids to glycerophosphoesters by dealkylation with boron trichloride: a tool for structural analysis of archaebacterial lipids

A method for preparing glycerophosphoesters from ether phospholipids by dealkylation with boron trichloride (BCl3) is described. Treatment of ether phospholipids in chloroform with BCl3 for 30 min at room temperature yielded almost quantitatively the corresponding glycerophosphoesters retaining the intact polar head group of the ether phospholipids. Thus, glycerophosphocholine, glycerophosphoinositol, glycerophosphoglycerol, glycerophosphoserine, glycerophosphate, and glycerophosphoethanolamine were prepared from the diether analogs of phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, and phosphatidic acid, and the tetraether analog of phosphatidylethanolamine, respectively. BCl3 also cleaved diacyl, alkyl-acyl, and alk-1-enyl-acyl forms of phospholipids to yield corresponding glycerophosphoesters. The glycerophosphoesters were separated more rapidly by cellulose thin-layer chromatography with the same solvent system as in paper chromatography. This method is of great use for structure determination of glycerophosphoester backbones of ether phospholipids, analogous to the mild alkaline methanolysis of diacyl form of phospholipids, as well as for the analysis of alkyl chains. It is, however, not applicable to glycolipids because of cleavage of glycosidic bonds by BCl3.     

Synthesis and activity of a novel diether phosphonoglycerol in phospholipase-resistant synthetic lipid:peptide lung surfactants()

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf?) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.     

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.     

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.     

Effect of the asymmetric Ca2+ distribution on the bilayer properties of phosphatidylcholine-sonicated vesicles

The incorporation of Ca2+ in the inner volume of egg phosphatidylcholine vesicles increases the fluorescence anisotropy of a diphenylhexatriene probe. This increase is higher than for Na+ at the same normality. An effect of the same magnitude is induced by Ca2+ when using binary lipid mixture (dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine) as long as the mixture is maintained below the phase-transition temperature of the saturated species. The influence of Ca2+ may be explained by an asymmetric distribution of the saturated and unsaturated lipids between the internal and the external monolayers.     

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.     

Effect of tryptophan derivatives on the phase properties of bilayers

Binding of several tryptophan derivatives and tryptophan-containing peptides to bilayers is examined by monitoring fluorescence enhancement as a function of lipid concentration. The thermodynamic and spectral parameters of the solutes in the bilayers of vesicles and liposomes do not exhibit any anomalous dependence upon the gel or the liquid-crystalline phase state of the bilayer. Effects of these solutes on the phase-transition profiles of the bilayers of liposomes and vesicles are examined, and the lowering of the phase-transition temperature is correlated with the mole fraction of the solute in the bilayer. The partition coefficients do not change at the main phase-transition temperature. These observations contradict the thermodynamic explanation of the solute-induced lowering of the phase-transition temperature which is based on the Van't Hoff relationship for distribution of the solute in the two coexisting phases at the phase-transition temperature. It is postulated that solute molecules bound to defect sites in bilayers modulate the phase properties of bilayers. These defect sites are induced in the gel phase of bilayers of liposomes above the subtransition temperature.     

Characterization and identification of glyceryl ether diesters present in tumor cells

The previously unidentified neutral lipid present in tumor tissues has been isolated from Ehrlich ascites cells and unequivocally identified as a lipid class of glyceryl ether diesters containing various degrees of unsaturation, and ranging in approximate molecular weight from 760 to 990. The glyceryl ether diester fraction was shown to be free from neutral plasmalogens (glyceryl diacyl alk-1'-enyl ethers). The tumor lipid was subjected to saponification, transesterification, and lithium aluminum hydride reduction. The glyceryl monoethers that resulted from deacylation were the 1-isomers ranging in hydrocarbon chain length from C(12) to C(24). The predominant glyceryl ethers were the hexadecyl (49%), octadecyl (21%), and octadecenyl (14%) derivatives. Saturated and mono- and polyunsaturated fatty acids ranging in chain length from C(12) to C(24) carbon atoms were esterified to the glyceryl monoether. Gas-liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance and infrared spectroscopy were used to characterize and identify the intact tumor lipid and its derived products.