Main Phospholipids Research Articles

High sensitivity differential scanning calorimetry investigation of the interaction between liposomes, lactate dehydrogenase and tyrosinase

High sensitivity differential scanning calorimetry (HSDSC) has been used to study the interaction of the model proteins lactate dehydrogenase (LDH) and tyrosinase with dimyristoylphosphatidylcholine (DMPC) liposomes, and relate this to the thermal and physical stability of the proteins. On heating, both LDH and tyrosinase denatured irreversibly in a time-dependent manner and modified the phase transition behaviour of DMPC liposomes at all concentrations investigated. The most marked effects occurred for the pretransition rather than the main phospholipid phase transition. The effects on the bilayer are likely to result from electrostatic interactions of the hydrophilic proteins with the head-groups of DMPC molecules, whilst due to their hydrophilic nature they do not penetrate into the bilayer. Tyrosinase is more highly ionised than LDH at the pH of the investigation, which may explain why tyrosinase has a greater effect than LDH on the HSDSC scans at mg/ml protein concentrations.

Saposin B binds and transfers phospholipids

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.

Analysis of phospholipid species in rat peritoneal surface layer by liquid chromatography/electrospray ionization ion-trap mass spectrometry

The main phospholipids in rat peritoneal surface layer were analyzed by normal-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion-trap mass spectrometry (MS). By using a silica gel column and a gradient of hexane/isopropanol/water as mobile phase containing 5 mmol/L ammonium formate as modifiers, a baseline separation of glycerophosphoehtanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and lyso-phosphatidylcholine (LPC) was obtained and more than 90 phospholipid constituents in rat peritoneal surface were identified and determined by on-line ion-trap MS detection. The major ethanolamine glycerophospholipids in rat peritoneal surfaces were plasmalogens that were highly enriched in polyunsaturated fatty acids at the sn-2 position. In addition, the fragmentation patterns for each phospholipid class by the ion-trap MS were discussed.

Endonuclear lipids in liver cellsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Lipids are not only components of cell nucleus membranes, but are also found in the membrane-depleted nuclei where they fulfill special functions. We have investigated the lipid composition of membrane-depleted rat liver nuclei obtained by incubation with low Triton X-100 concentrations of 0.04% and 0.08%, which rendered them unaltered or hardly altered. Under these conditions, 26% of proteins and 22% of phospholipids were recovered. The main phospholipids were phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol = or > phosphatidylserine and sphingomyelin (in decreasing concentrations). The fatty acid components of total lipids and phosphatidylcholine were mainly unsaturated. Over 40% belonged to the n-6 series (arachidonic > or = 25% and linoleic 15%); approximately 40% corresponded to saturated acids and <10% were monoenoic. Endonuclear phosphatidylcholine was built up by 16 molecular species, the most abundant being 18:0-20:4 (32%), 16:0-20:4 (19%), 16:0-18:2 (13%), and 18:0-18:2 (11%). The fatty acid composition and phosphatidylcholine molecular species distribution in the membrane-depleted nucleus of rat liver showed patterns similar to the whole nucleus, mitochondria, microsomes, and homogenate of the parent liver cells, suggesting that endonuclear lipid pool composition is mainly determined by a liver organ profile.

Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster)

The total lipids of muscle and cephalothorax of Mediterranean lobster Palinurus vulgaris were found to be 1.0% and 2.4% of the wet tissue of which the phospholipids represented 66.5% and 47.5%, respectively. The main PhL saturated, monounsaturated and polyunsaturated fatty acids in muscle and cephalothorax were C16:0, C18:0, C18:1ω-9, C18:1ω-7, C20:4ω-6, C20:5ω-3 and C22:6ω-3. 2-OH C14:0 and cyclo-17:0 fatty acids were also identified though in low percentages. The main individual PhL in muscle were found to be phosphatidylcholine (53.5%), 72.0% of which corresponded to the structure of 1,2-diacyl-glycerocholine while the rest 28.0% to 1- O-alkyl-2-acyl-glycerocholine or 1- O-(1-alkenyl)-2-acyl-glycerocholine and phosphatidylethanolamine (19.3%), 75.0% of which corresponded to the structure of 1,2-diacyl-glyceroethanolamine and 25% to 1- O-alkyl-2-acyl-glyceroethanolamine or 1- O-(1-alkenyl)-2-acyl-glyceroethanolamine. Cephalothorax main PhL were found to be PC and PE (66.4% and 18.8%, respectively). In muscle and cephalothorax PC ω-3 fatty acids amounted 7.78% and 8.60%, while in PE amounted 30.77% and 23.65% respectively. Furthermore, in both tissues PhL, cardiolipine phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatidylcholine, were also found.

Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) at the air–water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 °C and 24 °C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 °C and 37 °C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.

What can calorimetry tell us about changes of three-dimensional aggregate structures of phospholipids and glycolipids?

It is reported on the structural polymorphism of the main amphiphilic cell membrane compounds, phospholipids and glycolipids, with special regard to calorimetric analyses. These lipids may form a large variety of aggregate structures in dependence on their chemical primary structure, on temperature, water content, and concentrations of cations. The entity of aggregate structures is usually called the phase diagram of the respective lipids. This should, however, not to be confused with the gel and liquid crystalline phases of the hydrocarbon chains of lipids, which differ in the fluidity of the acyl chains, and between which a first order transition can be observed. Thus, in this contribution, exemplary results are presented on the structural behaviour of biologically important lipids including their phase behaviour and structural preferences under different ambient conditions, and how phase and structural transitions are connected with enthalpy changes.

Crystallization of cocoa butter with and without polar lipids evaluated by rheometry, calorimetry and polarized light microscopy

AbstractIn this investigation, we evaluated the crystallization process of triacylglycerols (TAG) in unrefined cocoa butter (CB) and in CB without polar lipids (CB‐WPL), under non‐isothermal and isothermal conditions. These conditions were obtained by cooling from 80 °C at a cooling rate of 1 °C/min until achieving particular crystallization temperatures (i.e., 18.5 °C, 19.0 °C, 19.5 °C, and 20 °C), at which isothermal crystallization studies were done. Phase shift angle rheograms (δ) showed that the onset of crystallization during the non‐isothermal stage, independently of the crystallization temperature (TCr) used, was 59.8 ± 3.7 °C in CB and 39.5 ± 1.5 °C in CB‐WPL. These results pointed out the nucleating role of phosphatidylcholine (26.53 ± 0.04%) and phosphatidylethanolamine (57.14 ± 0.07%), the main phospholipids present in the CB used in this investigation. Under similar crystallization conditions, differential scanning calorimetry (DSC) provided an onset of crystallization during the non‐isothermal stage of 15.0 ± 0.0 °C in CB‐WPL and of 15.5 ± 0.1 °C in CB. Then, δ rheograms were more sensitive to detect the nucleating role of phospholipids than DSC. Under isothermal conditions, both DSC and δ rheograms showed that shorter times were needed in CB‐WPL to complete crystallization than in CB. In the same way, at all TCr investigated, a higher crystallization rate was achieved in CB‐WPL than in CB, this as measured by the crystallization rate constant (z) of the Avrami equation. These results showed that crystallization of α and β' polymorphs took longer in CB than in CB‐WPL, pointing out that polar lipids delayed the α‐to‐β' polymorphic transition. Additional results confirmed that polar lipids affect the kinetics of TAG crystallization in CB. However, polar lipids do not affect the thermodynamic properties of the crystals [i.e., heat of fusion (δHM), temperature of fusion (TM')], the storage modulus at the end of the crystallization process (i.e., G' of pseudo‐equilibrium), or the mechanism of crystal growth (i.e., the Avrami index, n).

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

Lipid composition of the main fractions of rabbit semen

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 μg/10 9 but was only 21–23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp p) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE + PC ratios than in the germinal cells. Some physiological implications are discussed.

Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

The long-term response of the broad-salt growing halophile Chromohalobacter salexigens DSM 3043 T to salt stress has been investigated with respect to adaptive changes in membrane lipid composition. This study included the wild-type and three salt-sensitive, ectoine-deficient strains: CHR62 ( ectA::Tn 1732, unable to grow above 0.75 M NaCl), CHR63 ( ectC::Tn 1732, unable to grow above 1.5 M NaCl), and CHR64, which was able to grow in minimal medium M63 up to 2.5 M NaCl, but its growth was slower than the wild-type strain at salinities above 1.5 M NaCl. This mutant accumulated ectoine and hydroxyectoine as major compatible solutes, but also the ectoine precursor, N- γ-acetyldiaminobutyric acid, and was found to be affected in the ectoine synthase gene ectC. The main phospholipids of the wild-type strain were phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL). Major fatty acids were detected as 16:0, 18:1, and 16:1, including significant amounts of cyc-19:0, and cyc-17:0. CL and cyclopropane fatty acids (CFA) levels were elevated when the wild-type strain was grown at high salinity (2.5 M NaCl). Membranes of the most salt-sensitive trains CHR62 and CHR63, but not of the less salt-sensitive strain CHR64, contained lower levels of CL. The proportion of cyc-19:0 in CHR64 was three-fold (at 2.0 M NaCl) and 2.5-fold (at 2.5 M NaCl) lower than that of the wild type, suggesting that this mutant has a limited capacity to incorporate CFA into phospholipids at high salt. The addition of 1 mM ectoine to cultures of the wild-type strain increased the ratio PG/CL from 1.8 to 3.3 at 0.75 M NaCl, and from 1 to 6.5 at 2.5 M NaCl, and led to a slight decrease in CFA content. Addition of 1 mM ectoine to the mutants restored the steady-state levels of CL and CFA found in the wild-type strain supplemented with ectoine. These findings suggest that exogenous ectoine might attenuate the osmostress response involving changes in membrane lipids.

LIPID CHANGES IN FROZEN STORED FILLETS FROM PRE- AND POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI)

The lipid composition of frozen stored fillets from pre- and postspawned hake was studied. The total lipid (TL) content in the chloroform/methanol extract from unfrozen postspawned hake was four times higher than that of prespawned fish. After freezing, the TL content of postspawning hake muscle remained unchanged whereas the TL extracted from prespawning fish muscle increased about 90%. The TL extractability of muscle from fish in both different gonadal conditions was not affected by frozen storage. Lipolysis in frozen stored fillets from prespawned hake occurs principally by hydrolytic action on phospholipids (PL), and phosphatidylcholine was the main PL hydrolyzed. Triacylglycerols were the main substrates hydrolyzed in frozen stored fillets from postspawned hake. Freezing and frozen storage affected polyenoics and n-3 fatty acids (FA). The decrease in the contents of n-3 FA in fillets from postspawned hake was lower than that observed in fillets from prespawned fish.

The Phosphatidylglycerol/Cardiolipin Biosynthetic Pathway Is Required for the Activation of Inositol Phosphosphingolipid Phospholipase C, Isc1p, during Growth of Saccharomyces cerevisiae

Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537-11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Delta strains resembled that of pgs1Delta strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Delta strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262-25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed.

Modifying Calf Lung Surfactant by Hexadecanol

Monolayer properties such as phase behavior, collapse pressure, and surface viscosity are determined by monolayer composition. Learning how to control these properties through simple additives is important to understanding lung surfactant monolayers and to designing optimal replacement surfactants for treatment of disease. The properties of Infasurf, a replacement lung surfactant derived from calf lung lavage organic extract, can be modified in a controlled fashion by adding hexadecanol (HD). Grazing incidence X-ray diffraction shows that the HD preferentially interacts with dipalmitoylphosphatidylcholine (DPPC), the main phospholipid component of Infasurf, in the same way as in binary mixtures of DPPC and HD. HD intercalates between the DPPC chains, which leads to a tighter packing of the two-dimensional lattice and greater stability of the solid phase. This molecular reorganization triggers changes at the macroscopic scale, leading to a greatly increased solid-phase fraction at a given surface pressure and order of magnitude increases in the surface shear viscosity. However, the collapse pressure decreases, and, hence, the minimum surface tension of the film increases.

Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0-120 g l(-1), with the optimum at 10-20 g l(-1). The temperature range for growth at pH 7.0 was 4-50 degrees C, with the optimum at 37-40 degrees C. The optimum pH for growth was 7.0-8.0. The optimum pressure for growth was 0.1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2(T) (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA-DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

Intrinsic Structural and Functional Determinants within the Amino Acid Sequence of Mature Pulmonary Surfactant Protein SP-B

Pulmonary surfactant protein SP-B is absolutely required for proper function of surfactant in the alveoli, and is an important component of therapeutical surfactant preparations used to treat respiratory pathologies. To explore inherent structural and functional determinants within the amino acid sequence of mature SP-B, porcine SP-B has been subjected to extensive disulfide reduction under highly denaturing conditions and to cysteine carboxyamidomethylation, and the structure, lipid-protein interactions, and surface activity of this modified form have been characterized. Refolding of the reduced protein yielded a form (SP-Br) with secondary structure practically identical to that of the native disulfide-linked SP-B dimer. Reduced SP-Br exhibited higher structural flexibility than native SP-B, as indicated by a higher susceptibility of fluorescence emission to quenching by acrylamide and biphasic behavior during interaction of the protein with lipid bilayers and monolayers. SP-Br had, however, effects similar to those of native SP-B on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers. SP-Br was more effective than native SP-B in promoting interfacial adsorption of phospholipid bilayers into interfacial films, presumably because of its higher structural flexibility, and retained the ability of native SP-B to stabilize DPPC interfacial films compressed to pressures near collapse against spontaneous relaxation. SP-Br also mimicked the behavior of native SP-B in lipid-protein films subjected to dynamic compression-expansion cycling in a captive bubble surfactometer, but only in the presence of phosphatidylglycerol (PG), the main anionic phospholipid in surfactant. The presence of PG appears to be required for SP-Br to acquire the appropriate tertiary folding to produce progressively more efficient lipid-protein films capable of reaching very high pressures upon limited compression with almost no hysteresis.

Human Platelet Membrane Lipid Dynamics: Agonist Stimulation Results in the Aggregation of Lipid Rafts and the Lateral Phase Separation of like Lipids in Intact Human Platelets.

Sphingomyelin and cholesterol rich platelet membrane domains are organized into lipid rafts, which are present in the liquid ordered state, and which have been suggested to be key membrane components both during cold-induced human platelet activation (Gousset et al. 2002 Evidence for a physiological role for membrane rafts in human platelets. J. Cellular Physiol. 190:117–128) as well as during agonist induced activation. We have previous demonstrated that platelets have two membrane phase transitions, a raft transition at 34–40°C (Gousset et al., 2002 ) and a phospholipid phase transition at 10–20°C (Tablin et al. 1996 The membrane phase transition of intact human platelets: correlation with cold-induced activation. J. Cellular Physiol. 168:305–313). Using Fourier transform infrared spectroscopy (FTIR) we are able to sample all the key phases adopted by intact platelet phospholipids and sphingolipids (gel, liquid crystalline, liquid ordered (raft<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>)</INS>, hexagonal). This method offers unique advantages for the study of phospholipid acyl chain structures and interactions, as it is non-invasive and can detect molecular vibrations produced by dipole moment oscillations at infrared frequencies. Equally important<INS cite=mailto:John%20H%20Crowe dateTime=2004-04-08T16:45>,</INS> these vibrational frequencies are well characterized for individual phospholipids, permitting unambiguous assignment of the transitions to membranes in intact cells (Crowe et al. 1999. Are lipid phase transitions responsible for chilling damage in human platelets? Cryobiology 38:180-101). Most recently, using FTIR we have examined platelet lipid membrane dynamics associated with agonist induced human platelet activation. FTIR analysis of thrombin ( 1U/ml and 0.5U/ml) activated human platelets demonstrates that agonist stimulation results in the development of multiple (four or more) phase transitions, strongly suggestive of phase separation of lipids into like-lipid domains, also referred to as lateral phase separation. These like-lipid domains have phase transition temperatures very similar to that of isolated single lipid species, which, we have previously demonstrated combined to form the main phospholipids phase transition at between 10–20°C (Crowe et al. 1999). Furthermore, when human platelets are repeatedly scanned by FTIR - despite the temperature excursions which might normally result in the remixing of pure lipid populations, the thrombin treated platelets maintained their multiple phase transitions, suggesting that the lateral phase separation is an irreversible event during agonist induced activation. In addition to FTIR studies of human platelet populations, we have also examined the distribution of diIC18 in washed resting, and agonist stimulated human platelets. This dye, which preferentially partitions into liquid ordered or gel phases was uniformly distributed in resting platelets, but became aggregated when platelets were treated by either low concentrations of thrombin (0.01U/ml) or collagen (3.0ug/ml). In addition, platelets treated with higher concentrations of either agonist, demonstrated lipid rafts which further aggregated into larger liquid ordered domains. These results strongly suggest that platelet membrane lipid organization plays a key role in membrane function and further downstream signaling. Funded by NHLBI and DARPA.

A study of liposome formation using a solution (isoperibol) calorimeter

A solution (isoperibol) calorimeter has been employed to study the process of formation of phospholipid vesicles from natural and synthetic phospholipid films. Phospholipid films were hydrated in the solution calorimeter at temperatures exceeding the main phospholipid phase transition temperature, with continuous agitation to ensure conversion of the hydrating bilayers into multilamellar liposomes. It was seen that retention of chloroform in phospholipid films altered the apparent enthalpy change of vesicle formation to a far greater extent than would be expected from the contribution of the enthalpy of solution of chloroform; this indicates that chloroform alters the hydration process of the lipid. The overall measured enthalpy change for the formation of egg phosphatidylcholine vesicles was exothermic, whilst that for dimyristoylphosphatidylcholine was endothermic. This difference, it is suggested, results from the influence of the hydrocarbon chains mostly on the hydration process and also on the process of vesicle formation.

Structural identification of human blood phospholipids using liquid chromatography/quadrupole-linear ion trap mass spectrometry

A normal-phase liquid chromatography/quadrupole-linear ion trap mass spectrometry method was developed for the separation and mass spectral characterization of the main phospholipid species in human blood. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. The optimal separation was achieved by using hexane/1-propanol as mobile phase and 0.6% formic acid, 0.06% ammonia as modifiers. The HPLC/MS technique was able to provide information about the molecular mass of individual homologues by positive and negative turbo ionspray. More complete characterization of fatty acid chains and of the polar head group was obtained by using a quadrupole collisionally activated dissociation (CAD) spectrum with ion trap sensitivity. The mass spectra and molecular species of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and sphingomyelin (SM) are presented.

Antiphospholipid antibodies as cause of pregnancy loss.

Antiphospholipid antibodies detected by lupus anticoagulant, anticardiolipin or anti-beta2 glycoprotein I assays were associated with fetal loss. Rather than being diagnostic tools only, antiphospholipid antibodies are thought to be pathogenic. The strongest demonstration of their pathogenic role lies in the ability to induce fetal resorptions--the experimental equivalents of the human fetal losses--when passively infused in pregnant naive animals. However, still debated is how the antibodies might induce the obstetrical manifestations. Thrombotic events at the placental levels might be related to endothelial cell activation, inhibition of protein C/S system and fibrinolysis as well as to Annexin V displacement. However, the thrombophilic state apparently cannot explain all the miscarriages and a direct antibody-mediated damage on the trophoblast has been suggested. During differentiation to syncytium, trophoblasts express cell membrane anionic phospholipids that can bind beta2 glycoprotein I, the main cationic phospholipid binding protein recognized by the antiphospholipid antibodies. Adhered beta2-glycoprotein I might be recognized by the antibodies that, once bound, strongly interfere with in vitro trophoblast cell maturation so resulting in a defective placentation. These mechanisms have been suggested to play a role in early fetal loss, while thrombotic events would be responsible for miscarriages late in the pregnancy.