Solid- and liquid-phase equilibria in phosphatidylcholine/phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5 degrees C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mole fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847-854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Solid- and liquid-phase equilibria in phosphatidylcholine / phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5°C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mode fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847–854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: dodecanoic and 3-phenylpropionic acids system

Dimer structure and liquid structure of fatty acids in the binary liquid mixture of dodecanoic (LA) and 3-phenylpropionic acids (PPA) were studied through the measurements of DSC, self-diffusion coefficient (D), density, viscosity, 13C NMR spin-lattice relaxation time, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS). The phase diagram of LA/PPA mixture exhibited a typical eutectic pattern, which means that LA and PPA are completely immiscible in solid phase. In the liquid phase of the LA/PPA mixture, D of LA always differed from that of PPA irrespective of their compositions. This exhibited that, in the liquid phase of the binary mixture of fatty acids giving a complete eutectic in the solid phase, the fatty acid dimers are composed of the same fatty acid species irrespective of their compositions. The liquid structure of the LA/PPA mixture was clarified through the SAXS and also the SANS measurements.     

An index of lipid phase diagrams

There is a growing awareness of the utility of lipid phase behavior data in studies of membrane-related phenomena. Such miscibility information is commonly reported in the form of temperature-composition (T-C) phase diagrams. The current index is a conduit to the relevant literature. It lists lipid phase diagrams, their components and conditions of measurement, and complete bibliographic information. The main focus of the index is on lipids of membrane origin where water is the dispersing medium. However, it also includes records on acylglycerols, fatty acids, cationic lipids, and detergent-containing systems. The miscibility of synthetic and natural lipids with other lipids, with water, and with biomolecules (proteins, nucleic acids, carbohydrates, etc.) and non-biological materials (drugs, anesthetics, organic solvents, etc.) is within the purview of the index. There are 2188 phase diagram records in the index, the bulk (81%) of which refers to binary (two-component) T-C phase diagrams. The remainder is made up of more complex (ternary, quaternary) systems, pressure-T phase diagrams, and other more exotic miscibility studies. The index covers the period from 1965 through to July, 2001.     

Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: decanoic/octadecanoic acid and decanoic/dodecanoic acid systems

Dimer structure and liquid structure of fatty acids in their binary mixtures such as decanoic acid (DA)/octadecanoic acid (SA) and DA/dodecanoic acid (LA) were studied through the measurements of self-diffusion coefficient (D), differential scanning calorimetry (DSC), density and viscosity. The obtained phase diagrams showed that DA and SA form a eutectic in the solid state but partly a solid solution in the SA-rich region; DA and LA form an incongruent-melting compound which forms a eutectic with DA. In the liquid mixture of DA and SA, the D of DA is larger than that of SA over the entire range of compositions and tends to approach the D of SA with increasing SA-mole fraction; the D of DA in the DA/LA system is also larger than that of LA especially in the LA-poor region and steeply approaches that of LA with increasing LA-mole fraction. These D values and phase diagrams were compared with those for the binary mixtures of n-alkanes (C14/C20, C19/C20 and C20/C24); it is concluded that the two kinds of fatty acids always form their individual homodimers in their liquid mixtures regardless of their compositions and temperatures.     

The effects of topical application of various fatty acids on pheromone and glandular lipid biosynthesis in the moth Heliothis virescens

Binary mixtures of deuterium-labeled palmitic acid and an excess of different fatty acids were applied to the sex pheromone gland of female Heliothis virescens and the effects on the terminal steps of pheromone biosynthesis, including incorporation of fatty acids into the glandular lipids, observed. Relative to labeled palmitic acid applied alone, application of all the binary mixtures resulted in decreased levels of the labeled pheromone component, (Z)-11-hexadecenyl acetate (Z11-16:OAc), but there was generally no decrease in the amounts of labeled pheromone precursor, (Z)-11-hexadecenoate, nor labeled palmitate in the glandular lipids. These data suggest that the excess of fatty acid in the gland inhibits Delta11-desaturation. However, in the case of excess myristoleic acid, the amount of labeled (Z)-11-hexadecenoate increased significantly, suggesting that this acid inhibited fatty acid reduction. Dose-response tests with certain of the fatty acids were consistent with the above interpretations and further indicated that the gland had a high capacity for rapidly activating and incorporating excess fatty acids into the glandular lipids. Finally, application of the various fatty acids resulted in increased levels of these acids in the gland and, in the cases of myristoleic, palmitoleic and myristic acids, it also resulted in increased levels of the corresponding aldehydes, which had previously been detected in the gland of female H. virescens. This suggests that the fatty acid reductase in H. virescens is not highly specific for the major component, and that the final ratio of pheromone components is determined in part by the availability of their corresponding fatty acids in the gland.     

皱皮木瓜果实中有机酸成分的GC-MS分析

木瓜为常用中药,其原植物为木瓜属（Chaenomeles Lindl．）植物皱皮木瓜[Chaenomeles speciosa（Sweet）Nakai],又称贴梗海棠、贴梗木瓜等,中国南方各地均有栽培。其花可供观赏;果实入药,能驱风强壮、舒筋、镇痛消肿,民间常用于浸泡药酒;种仁富含油酸和亚油酸。对皱皮木瓜有机酸的脂肪性成分已有研究报道,但尚无其水溶性有机酸成分的研究报道。皱皮木瓜的药用功效主要为镇痛解痉,但至今未明确其主效成分,有机酸虽具有一定的镇痛功效,但有机酸的种类不同,功效也不相同。鉴于长期以来皱皮木瓜还是作为汤药服用,所以就其水溶性有机酸组成及含量开展研究,特别是与其有机酸中的脂肪性成分进行比较研究尤为必要,对于揭示皱皮木瓜主要功效成分具有重要的意义。     

Construction and Validation of Binary Phase Diagram for Amorphous Solid Dispersion Using Flory–Huggins Theory

Drug–polymer miscibility is one of the fundamental prerequisite for the successful design and development of amorphous solid dispersion formulation. The purpose of the present work is to provide an example of the theoretical estimation of drug–polymer miscibility and solubility on the basis of Flory–Huggins (F–H) theory and experimental validation of the phase diagram. The F–H interaction parameter, χ _d-p, of model system, aceclofenac and Soluplus, was estimated by two methods: by melting point depression of drug in presence of different polymer fractions and by Hildebrand and Scott solubility parameter calculations. The simplified relationship between the F–H interaction parameter and temperature was established. This enabled us to generate free energy of mixing (ΔG _mix) curves for varying drug–polymer compositions at different temperatures and finally the spinodal curve. The predicted behavior of the binary system was evaluated through X-ray diffraction, differential scanning calorimetry, and in vitro dissolution studies. The results suggest possibility of employing interaction parameter as preliminary tool for the estimation of drug–polymer miscibility.     

Optimising Drug Solubilisation in Amorphous Polymer Dispersions: Rational Selection of Hot-melt Extrusion Processing Parameters

The aim of this article was to construct a T–ϕ phase diagram for a model drug (FD) and amorphous polymer (Eudragit® EPO) and to use this information to understand the impact of how temperature–composition coordinates influenced the final properties of the extrudate. Defining process boundaries and understanding drug solubility in polymeric carriers is of utmost importance and will help in the successful manufacture of new delivery platforms for BCS class II drugs. Physically mixed felodipine (FD)–Eudragit^® EPO (EPO) binary mixtures with pre-determined weight fractions were analysed using DSC to measure the endset of melting and glass transition temperature. Extrudates of 10 wt% FD–EPO were processed using temperatures (110°C, 126°C, 140°C and 150°C) selected from the temperature–composition (T–ϕ) phase diagrams and processing screw speed of 20, 100 and 200rpm. Extrudates were characterised using powder X-ray diffraction (PXRD), optical, polarised light and Raman microscopy. To ensure formation of a binary amorphous drug dispersion (ADD) at a specific composition, HME processing temperatures should at least be equal to, or exceed, the corresponding temperature value on the liquid–solid curve in a F–H T–ϕ phase diagram. If extruded between the spinodal and liquid–solid curve, the lack of thermodynamic forces to attain complete drug amorphisation may be compensated for through the use of an increased screw speed. Constructing F–H T–ϕ phase diagrams are valuable not only in the understanding drug–polymer miscibility behaviour but also in rationalising the selection of important processing parameters for HME to ensure miscibility of drug and polymer.KEY WORDS: DSC, Flory–Huggins theory, hot-melt extrusion, thermal processing     

Synthesis and applications of stereospecifically (3)H-labeled arachidonic acids as mechanistic probes for lipoxygenase and cyclooxygenase catalysis

Stereospecifically (3)H-labeled substrates are useful tools in studying the mechanism of hydrogen abstractions involved in the oxygenation of polyunsaturated fatty acids. Here, we describe modified methods for the synthesis of arachidonic acids labeled with a single chiral tritium on the methylene groups at carbons 10 or 13. The appropriate starting material is a ketooctadecanoic acid which is prepared from an unsaturated C18 fatty acid precursor or by total synthesis. The (3)H label is introduced by NaB(3)H(4) reduction and the resulting tritiated hydroxy fatty acid then is tosylated, separated into the enantiomers by chiral phase HPLC, and subsequently transformed into stearic acids. A variety of stereospecifically labeled unsaturated fatty acids are obtained using literature methods of microbial transformation with the fungus Saprolegnia parasitica. Two applications are described: (i) In incubations of [10S-(3)H]- and [10R-(3)H]arachidonic acids in human psoriatic scales we show that a 12R-lipoxygenase accounts not only for synthesis of the major product 12R-HETE, but it contributes also, through subsequent isomerization, to the minor amounts of 12S-HETE. (ii) The [10R-(3)H]- and [10S-(3)H]arachidonic acids were also used to demonstrate that prostaglandin ring formation by cyclooxygenases does not involve carbocation formation at C-10 of arachidonic acid as was hypothesized recently.     

A synchrotron X-ray diffraction characterization of the structure of complexes formed between sphingomyelin and cerebroside

Specific lipid-lipid interactions are believed to be responsible for lateral domain formation in the lipid bilayer matrix of cell membranes. The miscibility of glucocerebroside and sphingomyelin extracted from biological tissues has been examined by synchrotron X-ray powder diffraction methods. Fully hydrated binary mixtures of egg-sphingomyelin codispersed with glucosylceramide rich in saturated C22 and C24 N-acyl fatty acids were subjected to heating scans between 20 and 90 °C at 2 °C·min(-1). X-ray scattering intensity profiles were recorded at 1 °C intervals simultaneously in both small-angle and wide-angle scattering regions. A gel phase characterized by a single symmetric peak in the wide-angle scattering region was transformed in all mixtures examined to a fluid phase at about 40 °C, similar to dispersions of pure egg-sphingomyelin. A coexisting lamellar structure was identified at temperatures up to about 75 °C which was characterized by a broad Bragg reflection. The scattering intensity of this structure increased relative to the structure assigned as bilayers of pure sphingomyelin with increasing proportions of glucosylceramide in the mixture. The relationship between the scattering intensities of the two peaks and the relative mass fractions of the two lipids showed that the bilayers assigned to a glucosylceramide-rich structure were composed of sphingomyelin and glucosylceramide in molar ratios of 1 : 1 and 2 : 1, respectively, at temperatures below and above the order-disorder phase transition temperature of the sphingomyelin (40 °C).     

Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites.

It is known that triphenylethylene anti-oestrogens such as tamoxifen bind to specific high-affinity anti-oestrogen-binding sites, which are distinct from oestrogen receptors. These binding sites are widely distributed in human and animal tissues, but their function and endogenous ligands are unknown. By using [3H]tamoxifen and a rat liver microsomal fraction, a radio-ligand-binding assay was developed in an attempt to identify endogenous ligands for the anti-oestrogen-binding sites in the rat. An ether extract of rat serum inhibited [3H]tamoxifen binding to rat liver binding sites in a dose-dependent manner. Identification of the active serum constituents that inhibited [3H]tamoxifen binding was achieved by g.l.c.-mass spectrometry after preliminary purification of a rat serum extract by silica-gel t.l.c. Three unsaturated fatty acids (oleic, linoleic and arachidonic) accounted for about 50% of the total inhibiting activity of the serum extract. The concentrations of these fatty acids required to inhibit [3H]tamoxifen binding were in the range of 10-100 microM, comparable with those found in the rat circulation under physiological conditions. Saturated fatty acids present in rat serum (palmitic and stearic) did not inhibit [3H]tamoxifen binding. A survey of other fatty acids revealed that, in general, unsaturated fatty acids were far more potent than saturated fatty acids in inhibiting [3H]tamoxifen binding. These studies demonstrate that unsaturated fatty acids are quantitatively the most important circulating inhibitors of [3H]tamoxifen binding to the anti-oestrogen-binding sites. The biological significance of their interaction with these sites, however, remains to be clarified.     

Miscibility and phase behavior of N-acylethanolamine/diacylphosphatidylethanolamine binary mixtures of matched acyl chainlengths (N=14, 16)

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and (31)P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by (31)P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with (31)P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65 degrees C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.     

Closed-loop miscibility gap and quantitative tie-lines in ternary membranes containing diphytanoyl PC

Vesicles containing ternary mixtures of diphytanoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and cholesterol produce coexisting liquid phases over an unusually large range of temperature and composition. Liquid domains persist well above the DPPC chain melting temperature (41 degrees C), resulting in a closed-loop miscibility gap bounded by two critical points at fixed temperature. Quantitative tie-lines are determined directly from 2H NMR spectra using a novel analysis, and are found to connect a liquid-disordered phase rich in diphytanoyl PC with a liquid-ordered phase rich in DPPC. The direction of the tie-lines implies that binary DPPC/cholesterol mixtures are in one uniform phase above 41 degrees C. All 2H NMR results for tie-lines are verified by independent fluorescence microscopy results.     

两种水华藻——球形棕囊藻和铜绿微囊藻的脂肪酸组成特征与水华形成机制

浮游植物所含的不饱和脂肪酸是测定其作为食物质量的指标,并在浮游植物向浮游动物及其它动物能量转化过程中起着关键的作用,必需不饱和脂肪酸的缺乏有利于水华的形成。球形棕囊藻(Phaeocystis globosa)和铜绿微囊藻(Microcystis aeruginosa)分别是常见的海洋和淡水水华藻类,该文分析了它们在不同生长期的脂肪酸组成,探讨了这两种藻类的脂肪酸组成特征。球形棕囊藻和铜绿微囊藻的脂肪酸碳链长为14~20个碳原子,脂肪酸种类组成都比较简单,以饱和脂肪酸为主,未检测到二十碳五烯酸(Eicosapentaenoic acid,EPA)和二十二碳六烯酸 (Docosahexaenoic acid,DHA)等动物的必需脂肪酸。球形棕囊藻的总脂肪酸含量在247.294~735.44 μg·g^-1干重之间,在对数期和延滞期含量最高的脂肪酸分别是C14:0和C16:0;而两株铜绿微囊藻的总脂肪酸在1 405.095~6 087.617μg·g^-1干重之间,以C16:0含量最高。两株铜绿微囊藻的脂肪酸含量在对数期和延滞期差异明显(p<0.05),但球形棕囊藻的脂肪酸含量在不同生长期差别不大。由于缺乏必需脂肪酸EPA和DHA,球形棕囊藻和铜绿微囊藻不能为高营养级的生物提供必需的不饱和脂肪酸,不是浮游动物等生物的良好食物。因此球形棕囊藻和铜绿微囊藻作为浮游动物的食物质量较低,浮游动物对它们的捕食压力也较小,可能是这两种藻容易暴发水华的重要原因。     

Biosynthetic incorporation of fatty acids with photosensitive groups into membrane lipids of cells in tissue culture.

The physical methods (13C-NMR-spectroscopy and fluorescence spectroscopy) hitherto used for the elucidation of lipid-lipid and lipid-protein interactions in artificial and simple natural membranes were extended to the application of fatty acids, phospholipids and sphingolipids with photochemical labels (azide group) in defined positions, which on photolysis generate nitrenes. These highly reactive groups react with neighbouring molecules, either lipids or polypeptide chains, with insertion or addition. Highly radioactive 12-azido[9,10-3H2]stearic acid, 12-azido[12-3H]oleic acid and 18-azido-[9,10,12,13-3H4]linoleic acid were added to the growth medium of eukaryotic cell lines in tissue culture (BHK 21 cells and Chang liver cells). They were incorporated into neutral, phosphoand sphingolipids in amounts comparable with the unsubstituted parent fatty acids. The distribution of the azido fatty acids in the phospholipids has been determined by enzymatic hydrolysis (phospholipase A2) on the basis of the distribution of their radioactivity. Radio gas chromatography and combined gas chromatography and mass spectroscopy revealed that the azide group of the radioactive fatty acids remained unaltered.     

Discrimination in resolving systems II: Ephedrine-substituted mandelic acids

Binary diastereomeric (-) (1R,2S)-ephedrine salts of various mandelic acids obtained from 95% ethanol show considerable differences in solubility. Structures and some properties of the less-soluble (L) and more-soluble (M) solid phases of (-)-ephedrine with unsubstituted mandelic acid, 2-, 3-, and 4-monosubstituted halo (F, Cl, Br) mandelic acids, and 3- and 4-methylmandelic acids have been determined. Salts were found to be binary, without solvent of crystallization, and composed of double-layered arrays of alternating anions and cations linked by H-bonds normal to the layers. H-bonding links charged donors and acceptors usually along a crystallographic 2-fold screw axis. A striking discrimination is evident in that the (2R)-mandelate salts typically display a compact four-atom chain as the H-bonding repeating unit [⁺N—H…O(—C^?—O)…H-N′, C₂¹(4)] while the (2S)-mandelate salts adopt a more dimensionally variable six-atom chain repeating unit [⁺N—H…O—C^?—O…H—N′, C₂²(6)]. Two distinct packing schemes display the shorter H-bonding chain of the (2R)-mandelates which always occurs with ephedrinium ions in the fully extended conformation. Slightly greater packing efficiency and H-bonding energies of the (2R)-mandelate salts correlates with increased fusion points, lower solubilities (95% ethanol), and higher heats of fusion relative to the phase adopted by their diastereoisomers. In contrast, (2S)-mandelate salts exhibit considerably more structural variability involving all three major ephedrinium conformations, and at least four distinct packing motifs. Mandelates with larger 3′-substituents (Cl, Br, methyl) show similar property discriminations, but these occur with an opposing trend, that is, between phases in which the less-soluble salts contain (2S)-mandelates. Salts with 2-bromomandelate do not show property disparities and their structures are dissimilar to the other phases. © 1995 Wiley-Liss, Inc.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Gel-state metastability and nature of the azeotropic points in mixtures of saturated phosphatidylcholines and fatty acids

The temperature-composition phase diagrams of dipalmitoylphosphatidylcholine (DPPC)/palmitic acid and distearoylphosphatidylcholine (DSPC)/stearic acid mixtures in excess water were recorded using high-sensitivity differential scanning calorimetry. New, slowly reversible phase transitions were found at 38° C in DPPC/palmitic acid mixtures at 0.4–0.9 mole fractions of palmitic acid and at 46° C in the DSCP/stearic acid binary. These transitions reveal gel-state metastability of the mixtures which is caused most probably by co-crystallization of the two lipids as it cannot be observed in the pure components. Both mixtures display azeotropic behavior at 2 fatty acids per 1 phospholipid. The physical reasons for such behavior have been analyzed theoretically in the framework of the Bragg-Williams and the UNIversal QUAsiChemical (UNIQUAC) approximations. This analysis shows that the azeotropic points in the phase diagrams are due to a combination of compound formation in the solid state and close to random mixing in the liquid state of the mixtures. UNIQUAC provides better fits to the experimental phase diagrams since it accounts also for the dimer-monomer character of the phospholipid/fatty acid mixtures. At fatty acid mole fractions greater than 0.65–0.7 the excess fatty acids phase separate from the compound phase. The stability of the compound phase domains at low fatty acid concentrations in relation to their possible physiological role has been discussed.