Extraction Of Phospholipids Research Articles

Analysis of the phospholipid profile of metaphase II mouse oocytes undergoing vitrification.

Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.

Effect of seed roasting on canolol, tocopherol, and phospholipid contents, Maillard type reactions, and oxidative stability of mustard and rapeseed oils.

This work was carried out to study the effect of roasting on different compositional parameters and oil oxidative stability of three Brassica species (Brassica juncea (BJ), B. juncea var. oriental (BJO), and Brassica napus (rapeseed, RS)). After 10 min of roasting at 165 °C, canolol contents of BJ, BJO, and RS oil reached 297.8, 171.6, and 808.5 μg/g, and the phospholipid phosphorus contents reached 453.6, 342.6, and 224.2 μg/g oil, respectively. The BJ and BJO seeds showed more prominent browning reactions than RS, due to the presence of higher amounts of reducing sugars, lysine, arginine and the occurrence of Maillard type browning reactions of phospholipids. The UV-visible spectra, fluorescence, and pyrrole content showed the presence of browning reaction products in the roasted seed oils. Roasting increased the oxidative stability of all varieties. Canolol formation could only partially explain such observations. Other roasting effects such as phospholipid extraction and Maillard type browning reaction products were also responsible for the increased stability.

Elucidation of phosphatidylcholine isomers using two dimensional liquid chromatography coupled in-line with ozonolysis mass spectrometry

In this study, two dimensional (2D) liquid chromatography (LC) was coupled in-line with ozonolysis-mass spectrometry (O3-MS) for the elucidation of phosphatidylcholine (PC) structures in phospholipid extracts. In O3-MS, PC molecules carried by mobile phase pass through a length of gas-permeable tubing surrounded by ozone vapor resulting in oxidative cleavage of double bonds. The characteristic aldehyde products are then directly analyzed by eletrospray ionization (ESI)-MS, allowing assignment of the double bond positions within the PC fatty acyl chains. The on-line 2D-LC method employs hydrophilic interaction chromatography as the first dimension to separate phospholipid classes and reversed phase chromatography in the second dimension to further separate PC molecular species. Thus, by combing the separation power of 2D-LC with in-line O3-MS (2D-LC/O3-MS), PC species in complex mixtures can be identified including the detailed structure of their two fatty acid chains. When the 2D-LC/O3-MS method was applied to a rat liver phospholipid extract, 19 PC molecular species were identified with fatty acid isomers unambiguously assigned.

Process optimization on the extraction of phospholipids with enzymolysis method from eggs of large yellow croaker

Process optimization on the extraction of phospholipids with enzymolysis method from eggs of large yellow croaker

Solid-phase extraction approach for phospholipids profiling by titania-coated silica microspheres prior to reversed-phase liquid chromatography-evaporative light scattering detection and tandem mass spectrometry analysis.

A novel strategy for selectively adsorbing phospholipids (PLs) on titania-coated silica core-shell microspheres (TiO2/SiO2) was developed. The TiO2/SiO2 microspheres were prepared through water-vapor-induced internal hydrolysis and then characterized by SEM, UV-vis spectroscopy, X-ray diffraction, and measurements of Brunauer-Emmett-Teller surface area. Analyses showed that the titania layer was uniformly distributed onto the surface of silica particles. The TiO2/SiO2 microspheres were employed as sorbent in solid-phase extraction (SPE), and their absorptive ability was investigated by reversed-phase liquid chromatography-evaporative light scattering detection (RPLC-ELSD). Important factors that affect the extraction, such as loading buffer, eluting buffer, and elution volume, were investigated in detail and optimized by using standard samples. Results reveal that the developed SPE approach had higher recoveries for PLs than that based on pure TiO2 particles. The proposed SPE method was used for extraction of PLs from serum and showed great potential for identifying more kinds of endogenous PL metabolites by ultra performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). The proposed SPE method with the composite sorbent was used to screen PLs from a biological matrix with high selectivity and efficiency. This approach is a promising method for selective extraction of PLs in lipidomics or phospholipidomics.

Erratum: Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets

Erratum: Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets

Extraction of Phospholipids from Structured Dry Egg Yolk

AbstractChicken egg yolk is a concentrated source of phospholipids (PL). Extracting egg PL with high efficiency is vital to the availability and economics of this high‐valued lipid product. In this study, two types of structured dry egg yolk materials, yolk flakes and pellets, were prepared. Two commonly used solvents, hexane and ethanol, were tested on the extraction of total yolk lipids including the PL. The PL fraction was obtained by the conventional cold acetone precipitation. The drum‐dried yolk flakes were shown to be an ideal starting material for total lipid and PL extraction. Anhydrous ethanol can extract almost all the neutral lipids and PL with little change to the individual components of the native PL. A PL product with a purity of more than 90 % and a yield of 99 % can be prepared using this method.

Effect of enzymatic hydrolysis on the extractability of phospholipids from leftover egg yolk using supercritical CO2

Supercritical carbon dioxide (SC-CO2) has been applied as a “green” solvent for phospholipids (PL) extraction from egg yolk but with limited success due to low extraction yield and purity. This study investigated a combination of proteolytic and lipolytic enzyme treatments on the extractability of PL using SC-CO2 from the leftover yolk after IgY extraction. The effects of enzymatic treatments on the recovery and purity of PC from leftover yolk using SC-CO2 in the presence of 8% (mole %) ethanol as co-solvent were studied at 48.3MPa, 70°C, and CO2 flow rate of 1l/min (at ambient conditions). The average recovery and purity of PC extracted from the cream obtained after enzymatic treatment were 94% and 57%, in comparison to 47% and 47%, and 40% and 75% for dry yolk and pellet, respectively. These results indicated that enzymatic treatment had a great impact on increasing the extractability of PC from egg yolk.

Lipidomic study of olive fruit and oil using TiO2 nanoparticle based matrix solid-phase dispersion and MALDI-TOF/MS

A matrix solid-phase dispersion (MSPD) procedure, using titanium dioxide (TiO2) nanoparticle (NP) as sorbent, combined with a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) method was developed for selective extraction, fast visualization and qualitative analysis of phospholipids from olive fruit and oil samples. The protocol was optimized by testing different sorbents and washing and eluting solvents. The results indicated that clean MS spectrum could be obtained when TiO2 NP was utilized as MSPD sorbent, acidified water (pH5) was used for washing the matrix and enhancing the binding ability, and chloroform and methanol (1/2, v/v) were selected as eluting solvent. In addition, the efficiency of this method was compared with that of the classic Bligh & Dyer method which produced high abundance peaks of glycerides and suppressed the signals of phospholipids. Based on the optimum conditions, olive fruit and oil samples were successfully analyzed in both positive and negative ion modes. The whole method demonstrated to be promising for selective separation of phospholipids from non-phospholipids, and has great potential in fast lipidomic fingerprinting of olive fruits and oils for quality control.

Improved sensitivity of sedimentary phospholipid analysis resulting from a novel extract cleanup strategy

The detection of intact phospholipids (PLs) in sedimentary extracts using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) is commonly limited by ion suppression caused by complex matrices; the effect typically cannot be satisfactorily reduced using traditional column chromatographic cleanup procedures. We therefore implemented a cleanup strategy recently introduced for serum and plasma samples and adapted it for sedimentary lipid extracts. It involves selective retention of PLs on a zirconia-coated silica phase, available as a HybridSPE®-Phospholipid cartridge. Compared with conventional silica gel liquid chromatography, the HybridSPE-based protocol strongly enhances the detection of PLs using LC–ESI-MS as a result of more effective removal of complex environmental matrices, with negligible PL loss. The strength of the protocol was demonstrated by analysis of marine sediment samples from various depths and of different total organic carbon (TOC) content, for which we observed 1.2–2.7 times more intense signals from sedimentary PLs compared with the untreated reference. This new cleanup protocol significantly improves PL fingerprinting of microbial communities in sediments.

Linking membrane physical properties and low temperature tolerance in arthropods

Maintenance of membrane fluidity is of crucial importance in ectotherms experiencing thermal changes. This maintenance has in ectotherms most often been indicated using indirect measures of biochemical changes of phospholipid membranes, which is then assumed to modulate the physico-chemical properties of the membrane. Here, we measure bending rigidity characterizing the membrane flexibility of re-constituted membrane vesicles to provide a more direct link between membrane physical characteristics and low temperature tolerance. Bending rigidity of lipid bilayers was measured in vitro using Giant Unilamellar Vesicles formed from phospholipid extracts of the springtail, Folsomia candida. The bending rigidity of these membranes decreased when exposed to 0.4vol% ethanol (0.23mM/L). Springtails exposed to ethanol for 24h significantly increased their cold shock tolerance. Thus, by chemically inducing decreased membrane rigidity, we have shown a direct link between the physico-chemical properties of the membranes and the capacity to tolerate low temperature in a chill-susceptible arthropod.

Optimization of a solid phase extraction method and hydrophilic interaction liquid chromatography coupled to mass spectrometry for the determination of phospholipids in virgin olive oil

Phospholipids belong to a polar lipid class of compounds and are particularly important as constituents of membrane of lipids in several food products. Nevertheless, phospholipids are poorly investigated as minor compounds in olive oil and in literature the phospholipid extraction has been essentially developed by liquid–liquid extraction (LLE). The aim of this work was to compare the effectiveness of a previous LLE extraction method with a direct separation and purification of olive oil phospholipids with two different solid phase extraction (SPE) sorbents (silica and diol). As far we are concerned this is the first application of SPE for the direct extraction of phospholipids in virgin olive oil. Furthermore, the extracts were analyzed by HPLC with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS) to obtain basic information useful for a possible identification of the single components. To this aim, a new separation method was established using a hydrophilic fused core liquid chromatography column (HILIC). The HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The SPE diol extraction and HPLC–ESI-qTOF-MS methodology have demonstrated to be very useful to determine the phospholipids in not filtered virgin olive oil and also in a veiled virgin olive oil subjected to a glyceridic crystallization due to a temperature decrease. In fact, it could be observed that crystallization caused a significant decrease in the phospholipid fraction.

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.

Lipidomic Fingerprint of Almonds (Prunus dulcis L. cv Nonpareil) Using TiO2Nanoparticle Based Matrix Solid-Phase Dispersion and MALDI-TOF/MS and Its Potential in Geographical Origin Verification

A matrix solid-phase dispersion (MSPD) procedure with titanium dioxide (TiO2) nanoparticles (NP) as sorbent was developed for the selective extraction of phospholipids from almond samples, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) was employed for analysis. A remarkable increase in the signals of phospholipid accompanied by a decrease in those of triacylglycerols and diacylglycerols was observed in the relevant mass spectra. The proposed method was applied to five batches of almonds originating from four geographical areas, whereas principal component analysis (PCA) was utilized to normalize the relative amounts of the identified phospholipid species. The results indicated that the lipidomic fingerprint of almonds was successfully established by the negative ion mode spectrum, and the ratio of m/z 833.6 to 835.6 as well as m/z 821.6 could be introduced as potential markers for the differentiation of the tested almonds with different geographical origins. The whole method is of great promise for selective separation of phospholipids from nonphospholipids, especially the glycerides, and superior in fast screening and characterization of phospholipids in almond samples.

Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets

Understanding how nanomaterials interact with cell membranes is related to how they cause cytotoxicity and is therefore critical for designing safer biomedical applications. Recently, graphene (a two-dimensional nanomaterial) was shown to have antibacterial activity on Escherichia coli, but its underlying molecular mechanisms remain unknown. Here we show experimentally and theoretically that pristine graphene and graphene oxide nanosheets can induce the degradation of the inner and outer cell membranes of Escherichia coli, and reduce their viability. Transmission electron microscopy shows three rough stages, and molecular dynamics simulations reveal the atomic details of the process. Graphene nanosheets can penetrate into and extract large amounts of phospholipids from the cell membranes because of the strong dispersion interactions between graphene and lipid molecules. This destructive extraction offers a novel mechanism for the molecular basis of graphene's cytotoxicity and antibacterial activity.

Identification of Phospholipid Classes in Human Cancer and Normal Tissue Extracts by Nano-ESI Tandem Mass Spectrometry

Phospholipids can be classified into glycerol- and sphingo-phospholipids and into classes based on their polar head groups. The major glycerophospholipid classes are phosphatidylethanolamine (PE), phosphatidylcholine, phosphatylserine (PS) and phosphatidylinisitol (PI). In this study, total lipid and phospholipid extraction was performed according to the Bligh and Dyer method [1]. The extracts were analysed in both positive and negative ion modes ESI-MS for monitoring the present of intact phospholipids. Structural characterizations of each intact phospholipid were done in both positive and negative ion mode by electrospray ionization tandem-mass spectrometry (ESI-MS/MS). The MS/MS analyses of each protonated [M+H] + and deprotonated [M-H] molecule were performed at various different collision off set values in order to obtain the intense characteristic fragment ions in the MS/MS spectra. PC was identified in positive ion mode for all extracts due to their fixed positive charged at the choline group [2, 3] PS was able to be detected in positive and negative ion mode. In the extracts protonated PS at m/z 790 was detected at with a prominent fragment ion m/z 605. The PS species observed at m/z 790 is assigned as PS (18:0,18:1). In negative ion mode deprotonated singly charged ion and deprotonated doubly charged ion was observed at m/z 788.5 and at 393.8 respectively. PE, PI and plasmalogen-PE were also identified in the extracts. REFERENCES

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy.

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell-cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. 31P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in 31P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid 31P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible 31P NMR spectra of phospholipids.

Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom

The mechanism through which brown spiders (Loxosceles genus) cause dermonecrosis, dysregulated inflammatory responses, hemolysis and platelet aggregation, which are effects reported following spider bites, is currently attributed to the presence of phospholipase-D in the venom. In the present investigation, through two-dimensional immunoblotting, we observed immunological cross-reactivity for at least 25 spots in crude Loxosceles intermedia venom, indicating high expression levels for different isoforms of phospholipase-D. Using a recombinant phospholipase-D from the venom gland of L. intermedia (LiRecDT1) in phospholipid-degrading kinetic experiments, we determined that this phospholipase-D mainly hydrolyzes synthetic sphingomyelin in a time-dependent manner, generating ceramide 1-phosphate plus choline, as well as lysophosphatidylcholine, generating lysophosphatidic acid plus choline, but exhibits little activity against phosphatidylcholine. Through immunofluorescence assays with antibodies against LiRecDT1 and using a recombinant GFP-LiRecDT1 fusion protein, we observed direct binding of LiRecDT1 to the membrane of B16-F10 cells. We determined that LiRecDT1 hydrolyzes phospholipids in detergent extracts and from ghosts of B16-F10 cells, generating choline, indicating that the enzyme can access and modulate and has activity against membrane phospholipids. Additionally, using Fluo-4, a calcium-sensitive fluorophore, it was shown that treatment of cells with phospholipase-D induced an increase in the calcium concentration in the cytoplasm, but without altering viability or causing damage to cells. Finally, based on the known endogenous activity of phospholipase-D as an inducer of cell proliferation and the fact that LiRecDT1 binds to the cell surface, hydrolyzing phospholipids to generate bioactive lipids, we employed LiRecDT1 as an exogenous source of phospholipase-D in B16-F10 cells. Treatment of the cells was effective in increasing their proliferation in a time- and concentration-dependent manner, especially in the presence of synthetic sphingomyelin in the medium. The results described herein indicate the ability of brown spider phospholipase-D to induce the generation of bioactive phospholipids, calcium influx into the cytoplasm and cell proliferation, suggesting that this molecule can be used as a bioactive tool for experimental protocols in cell biology.

Comparative Analysis of Sample Preparation Methods To Handle the Complexity of the Blood Fluid Metabolome: When Less Is More

Blood sample preparation before LC-MS metabolomic fingerprinting is one of the most challenging and error-prone parts of the analytical procedure. Besides proteins, phospholipids contained in blood fluids are known to cause matrix effects and ion suppression phenomena, thus masking biological variation. Nevertheless, the commonly used sample preparation techniques do not consider their removal prior to analysis. Pooled plasma and serum samples were used as biological material, partly as raw samples and partly spiked with distinct concentrations of a metabolite mix (1-5 μg/mL). Prior to LC-ESI-qToF-MS-driven metabolomic analysis, samples were subjected to different preparation methods consisting of three extractions with organic solvents (acetonitrile, methanol, and methanol/ethanol), a membrane-based solvent-free technique, and a hybrid method combining solvent extraction and SPE-mediated removal of phospholipids. The comparative analysis among sample preparation procedures was based on the capacity to detect endogenous compounds in raw samples, differentiate raw versus spiked samples, and reveal real-life metabolomic changes, following a dietary intervention. Method speed, minimum sample handling, compatibility to automation, and applicability to large-scale metabolomic studies were also considered. The combination of solvent deproteinization and the selective removal of phospholipids was revealed to be the most suitable method, in terms of improvement of nonlipid metabolite coverage, extraction reproducibility, quickness, and compatibility with automation, the minimization of matrix effects being among the most probable causes for the good extraction performance associated with the removal of phospholipid species. The main advantage of conventional solvent extraction procedures was the metabolite information coverage for lipid low-molecular-weight species, and extraction with acetonitrile was generally the second choice for sample preparation. Ultrafiltration was the least effective method for plasma and serum preparation; thus, its use without a previous solvent extraction step of the samples should be discarded. According to the presented data, there is no apparent reason to believe that sacrificing information on lipid compounds is too high of a price to pay in order to gain more information on nonlipid LMW metabolites.

Adjusting membrane lipids under salt stress: the case of the moderate halophilic organism Halobacillus halophilus

The lipid composition of Halobacillus halophilus was investigated by combined thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the total lipid extract. Main polar lipids were found to be sulfoquinovosyldiacylglycerol and phosphatidylglycerol, while cardiolipin was a minor lipid together with phosphatidic acid, alanyl-phosphatidylglycerol and two not yet fully identified lipid components. In addition the analyses of residual lipids, associated with denatured proteins after the lipid extraction, revealed the presence of significant amounts of cardiolipin, indicating that it is a not readily extractable phospholipid. Post decay source mass spectrometry analyses allowed the determination of acyl chains of main lipid components. On increasing the culture medium salinity, an increase in the shorter chains and the presence of chain unsaturations were observed. These changes in the lipid core structures might compensate for the increase in packing and rigidity of phospholipid and sulfoglycolipid polar heads in high-salt medium, therefore contributing to the homeostasis of membrane fluidity and permeability in salt stress conditions.