Different Classes Of Phospholipids Research Articles

Spatial Analysis of Phospholipids in Insect Models by Positive Ionization Mode Matrix-Assisted Laser Desorption Ionization Mass Spectrometric Imaging

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging (MALDI-MSI) has developed as a useful tool in generating comprehensive metabolite profiles along with spatial information in model organisms. The ability of MALDI-MSI to generate in situ profiles in whole organisms or specific tissue sections serves as an efficient alternative to solvent extraction protocols, especially for the identification of biomolecules that are unstable under extraction conditions. Herein, we have utilized MALDI-MSI to spatially profile various classes of lipids in two model organisms, Drosophila melanogaster (fruit fly), and Tribolium castaneum (red flour beetle). Five different classes of phospholipids were imaged in positive ionization mode in both insect species. Confirmation of the m/z assignment for selected lipids was performed using on-tissue MS/MS fragmentation.

2D HILIC‐ELSD/UPLC‐Q‐TOF‐MS Method for Acquiring Phospholipid Profiles and the Application in Caenorhabditis elegans

AbstractPhospholipids are the main constituent of cellular membranes and have recently been identified to have diagnostic value as biomarkers for many diseases. Accordingly, much emphasis is now laid on developing optimal analytical techniques for the phospholipid profiles of various biological samples. In the present study, different classes of phospholipids are first separated by optimized hydrophilic interaction chromatography with evaporative light scattering detector (HILIC‐ELSD). The phospholipids in each class are then identified by ultraperformance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). Validation results confirm that this approach meets the requirements of quantitative analysis. Finally, the approach is adopted to analyze the phospholipid profiles in Caenorhabditis elegans. A total of 111 phospholipid species is identified according to the mass fragments. Major fatty acyl chains in phospholipids are found to be formed by oleic acid (C18:1), arachidonic acid (C20:4), and eicosapentaenoic acid (C20:5). Overall, this study improves current knowledge on analytical techniques of the phospholipid composition in C. elegans and provides a basis for future lipidomics research.Practical applications: Phospholipids reportedly play a crucial role in the development of many diseases. Until now, only a small portion of phospholipids in Caenorhabditis elegans has been reported by using one‐dimensional analysis strategy. The offline 2D liquid chromatography method developed in this study identifies 111 phospholipid species in Caenorhabditis elegans. The obtained phospholipid profiles complement the lipid database of Caenorhabditis elegans. The study also provides the basis for the future development of a 2D online approach.

Identification of unusual phospholipids from bovine heart mitochondria by HPLC-MS/MS

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Comprehensive Characterization of Phospholipid Isomers in Human Platelets

Lipids play an important role in platelet integrity and function. However, most of lipid molecular compositions in platelets still remain unknown, especially for phospholipids. In this work, we characterized detailed structures of different classes of phospholipids in human platelets. Using a flow microreactor, the acetone Paterno-Buchi reaction was introduced for online derivatization of carbon–carbon double bond (C=C) of lipids right after separation by liquid chromatography (LC). Tandem mass spectrometry (MS/MS) methods were developed for multi-level structural identification of phospholipids, including head group, chain composition, and C=C locations in the fatty acyl/alkyl chains. After optimization of the workflow, a total of 110 molecular species of phospholipids were identified in the human platelet samples, including many C=C location isomers. At lipid class level, phosphatidylserine was found more abundant in the platelet samples than that of the plasma samples; at isomer level, the relative ratio of C18:1 (Δ9) vs C18:1 (Δ11) also increased in platelet samples comparing with that of the plasma samples. The detailed structural information of phospholipids and their C=C isomers ratios would be useful for investigation of potential functions of human platelets potentially associated with phospholipid isomers.

Quantitative lipidomic insights in the inhibitory response of Pichia stipitis to vanillin, 5-hydroxymethylfurfural, and acetic acid

To obtain a global view of the dynamic phospholipids in Pichia stipitis during the ethanol fermentation in the presence of three representative inhibitors (vanillin, 5-hydroxymethylfurfural, and acetic acid). Considerable efforts have been expended to elucidate the biochemical mechanisms of inhibitors interaction with phospholipids. In this study, a comparative lipidomic analysis was performed using liquid chromatography-mass/mass spectrometry (LC-MS/MS) on P. stipitis. Partial least squares-discriminate analysis (PLS-DA) was used to deal with the large quantity of data generated using the systematic methods. PLS-DA revealed that phosphatidylinositol (PI) (PI34:1, PI34:2 and PI34:6), phosphatidylserine (PS) (PS34:1 and PS34:2), phosphatidylethanolamine (PE) (PE34:1 and PE34:2), and phosphatidylcholine (PC) (PC34:1, PC34:2, and PC34:3) were the predominant biomarkers. Further analysis of different classes of phospholipids indicated that: (a) the samples from three combined inhibitors condition during the lag phase possessed the lowest PI/PS value 1.4%, (b) alterations in PC/PE ratios with changes in inhibitors were coincident with the changes in xylose utilization rates, and (c) the levels of unsaturated and the relatively long chain phospholipids increased in the inhibitor-plus condition. These findings suggest that regulation of membrane properties with inhibitors might offer a way of self-protection of yeast to inhibitors stress.

Development of a direct in-matrix extraction (DIME) protocol for MALDI-TOF-MS detection of glycated phospholipids in heat-treated food samples.

In foodstuffs, one of the main factors inducing modifications in phospholipids (PLs) structure is the heat treatment. Among PLs, only phosphatidylethanolamines and phosphatidylserines, due to their free amino group, can be involved in Maillard reaction and can form adducts with reducing sugars, besides other by-products called advanced glycation end-products. To date, glycated lipid products are less characterized in comparison to proteins. The aim of this work was to develop a novel, rapid and sensitive extraction protocol for the detection and characterization of modified PLs (glycated and oxidized) by means of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). At first, to investigate the formation of glycated and/or short chain by-products in different classes of PLs, representative standards were heated with or without sugar (lactose or glucose) and subjected to traditional lipid extraction methods as Bligh and Dyer and to the novel direct in matrix extraction (DIME) using 1,8-bis(dimethylamino)naphthalene as preconcentrating matrix. MALDI-MS analysis in negative ion mode allowed detecting glycation and oxidation products both on fatty acid and glucose moieties. Then, the procedure was successfully applied to different heat-treated and powdered samples (milk powders, pasteurized milk, ultra-high-temperature milk and soy flour) for the detection of modified PLs in complex foods. The currently developed DIME protocol could be a powerful tool for understanding lipid glycation also in biological samples.

A Comprehensive Study of Preferential Interaction of Cholesterol and its Fluorescent Analogs with Different Classes of Phospholipids

Cell membranes are composed of various glycerophospholipids and sphingolipids. Cholesterol is believed to interact with these phospholipids to modulate the fluidity of the membrane. We have introduced a novel approach to measure the preferential interaction of cholesterol with different classes of phospholipids having different head-groups and acyl-chain compositions. Phospholipids involved in the study are phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS) and sphingomyelin (SM). These phospholipid classes constitute the bulk of the membrane phospholipids. The acyl chain configurations of the saturated and unsaturated glycerophospholipids involved in this study are di 16:0 and 16:0(sn1)-18:1(sn2) respectively. Correspondingly, the N-linked acyl chain configurations in SMs are 16:0 and 18:1(Δ9-cis). Affinity of sterol analogs cholestatrienol and bodipy-cholesterol towards phospholipid bilayers is also studied to compare their affinities with cholesterol. Large unilamellar vesicle (LUV) systems with (sterol) donor vesicles and (sterol) acceptor vesicles are used. Acceptor LUVs are allowed to accept the sterol from donor vesicles, transfer being assisted by methyl-β-cyclodextrin. Diphenylhexatriene-phosphatidylcholine (DPHPC) was used as a fluorescent probe in cholesterol donor acceptor anisotropy experiments. We observed that cholesterol showed different affinity for phospholipids as compared to its fluorescent analogs. Affinity of cholesterol and the fluorescent analogs for different phospholipid bilayers depended not only on the head groups of the phospholipids but also on their acyl chain configuration.

Sphingomyelin is more sensitively detectable as a negative ion than phosphatidylcholine: a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric study using 9‐aminoacridine (9‐AA) as matrix

Phospholipids (PLs) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and imaging MS. Different classes of PLs are preferentially detectable either as positive or negative ions depending on the charges of their headgroups. Sphingomyelin (SM) and phosphatidylcholine (PC) occur in virtually all biological samples and both are assumed to be detectable with the same sensitivity (in the positive ion mode) because their headgroups are identical. We will show here that the detectabilities of PC and SM depend on the matrix used. In the presence of 2,5-dihydroxybenzoic acid (DHB) SM is more sensitively detectable in positive ion mode than PC while the use of 9-aminoacridine (9-AA) as matrix inverts the detectabilities. Our explanation is that the preferred generation of negative ions from SM if 9-AA is used as matrix results in a reduced yield of positive ions. It will also be shown that this is not only valid if a simplified model system is investigated, but also if, for instance, extracts from human erythrocytes are investigated. It will also be outlined that this finding is particularly important in the context of imaging studies where no previous separation of the lipids of interest can be performed.

Binary Matrix for MALDI Imaging Mass Spectrometry of Phospholipids in Both Ion Modes

Phospholipids (PLs) are the major building block molecules of cellular membranes. Their composition varies depending on cell types and cellular compartments. Thus, the information regarding PL distribution in tissue has important physiological and pathological significance. Recent developments in imaging mass spectrometry (IMS) have allowed complete mapping of the PL species on tissue. The IMS technique can detect different classes of PLs as well as their location information directly from tissue sections. PL head groups carry either positive and/or negative charges; therefore, IMS experiments must be conducted in both positive- and negative-ion mode to detect all types of phospholipids. Several conventional matrixes were applied on tissue for better identification. This study was conducted to enable appropriate matrix selection and optimized matrix preparation for IMS experiments in both ion modes that maximize PL identification from a single brain tissue section. The optimized matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) with a mixture of trifluoroacetic acid (TFA) and piperidine as ion pairing agents showed improved stability and consistency during both ion mode experiments and successfully identified >100 peaks of PLs determined by parent ion m/z value. Further tandem mass spectrometric analysis (MS/MS) was performed to those PLs that are anatomically important according to their distribution on rat brain tissue section.

Comparative lipidomic profiling of xylose‐metabolizing S. cerevisiae and its parental strain in different media reveals correlations between membrane lipids and fermentation capacity

Phospholipids (PLs) serve as the foundation for structure and function in most cell membranes. In order to reveal the correlations between PLs composition and fermentation performance of cells, a comparative lipidomics study was carried out using a recombinant xylose fermenting yeast strain Saccharomyces cerevisiae 424A(LNH-ST) and its parental strain 4124. Profiling of yeast lipids was performed using ultra performance liquid chromatography (UPLC)-MS/MS, leading to identification of 123 PL species. PL compositions were determined for both strains grown in rich medium (yeast extract peptone), limited medium (yeast nitrogen base), and ammonia fiber expansion pretreated corn stover hydrolysate. Principal component analysis of lipidomic data revealed that the PL profile for both strains varied significantly depending upon cultivating media composition. Further analysis of different classes of PLs revealed that the phosphatidylinositol/phosphatidylserine (PI/PS) ratio was closely related to cell growth rates. Both strains possessed higher phosphatidylcholine (PC) levels at an expense of phosphatidylethanolamine (PE) levels when entering stationary phase and the PC/PE ratios showed consistency with glucose utilization rates. Interestingly, PI synthesis lagged behind when available nutrients were limited, and PI levels were closely correlated with xylose metabolism.

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MS n by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/ z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.

Analysis of Phospholipids in Rat Brain Using Liquid Chromatography–Mass Spectrometry

The brain is a lipid-rich organ containing complex polar lipids including phospholipids (PLs) and sphingolipids. These lipids are involved in the structure and function of cell membranes in the brain. We developed a fast and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify five different classes of PLs [Choline glycerophospholipid (consists of phosphatidyl choline and plasmenyl choline in these samples), ethanolamine glycerophospholipid (consist of phosphatidyl ethanolamine and plasmenyl ethanolamine in these samples), phosphatidyl serine, phosphatidyl inositol, and sphingomyelin] in the brain tissues of 80-day-old Wistar rats. The PLs were extracted from rat brain using chloroform/methanol/water. After separation using a hydrophilic high performance liquid chromatography column, PL-class-specific fragmentation (head group identification) with a tandem mass spectrometer in positive ion mode was utilized to measure changes in the relative concentration of the five PL classes. The advantage of this approach was its improved specificity over previously reported LC-MS methods. The method had good repeatability (coefficient of variation 3-9%, excluding phosphatidyl inositol) and recovery (92-103%) and compared well with more laborious traditional methods.

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids -- phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd(2)Gro) -- and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd(2)Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd(2)Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd(2)Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids -- Ptd(2)Gro >> PtdSer > PtdIns -- underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes -- PtdCho and PtdEtn -- was detected. MS-studies revealed the presence of hydroxy-, hydroperoxy- as well as hydroxy-/hydroperoxy-species of Ptd(2)Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd(2)Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H(2)O(2), confirmed that molecular identities of the products formed were similar to the ones generated during STS-induced neuronal apoptosis. The temporal sequence of biomarkers of STS-induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd(2)Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd(2)Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.

Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity

All organisms consist of cells that are enclosed by a cell membrane containing bipolar lipids and proteins. Glycerophospholipids are important not only as structural and functional components of cellular membrane but also as precursors of various lipid mediators. Polyunsaturated fatty acids comprising arachidonic acid or eicosapentaenoic acid are located at sn-2 position, but not at sn-1 position of glycerophospholipids in an asymmetrical manner. In addition to the asymmetry, the membrane diversity is important for membrane fluidity and curvature. To explain the asymmetrical distribution of fatty acids, the rapid turnover of sn-2 position was proposed in 1958 by Lands [Lands WE (1958) Metabolism of glycerolipides: A comparison of lecithin and triglyceride synthesis. J Biol Chem 231:883-888]. However, the molecular mechanisms and biological significance of the asymmetry remained unknown. Here, we describe a putative enzyme superfamily consisting mainly of three gene families, which catalyzes the transfer of acyl-CoAs to lysophospholipids to produce different classes of phospholipids. Among them, we characterized three important enzymes with different substrate specificities and tissue distributions; one, termed lysophosphatidylcholine acyltransferase-3 (a mammalian homologue of Drosophila nessy critical for embryogenesis), prefers arachidonoyl-CoA, and the other two enzymes incorporate oleoyl-CoAs to lysophosphatidylethanolamine and lysophosphatidylserine. Thus, we propose that the membrane diversity is produced by the concerted and overlapped reactions with multiple enzymes that recognize both the polar head group of glycerophospholipids and various acyl-CoAs. Our findings constitute a critical milestone for our understanding about how membrane diversity and asymmetry are established and their biological significance.

Functional and Structural Characterization of Rhodopsin Oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

A shotgun tandem mass spectrometric analysis of phospholipids with normal‐phase and/or reverse‐phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.

NADPH Oxidase-dependent Oxidation and Externalization of Phosphatidylserine during Apoptosis in Me2SO-differentiated HL-60 Cells: ROLE IN PHAGOCYTIC CLEARANCE

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.

Body Temperature Alters the Lipid Composition of Pulmonary Surfactant in the LizardCtenophorus nuchalis

In any 24-h period the body temperature (Tb) of the central Australian agamid lizard, Ctenophorus nuchalis, may vary from 13 to 45 degrees C; the mean preferred Tb is 37 degrees C. We have analyzed surfactant-type lipids in lizards that underwent rapid changes in Tb from 37 degrees C to 14, 19, 27, or 44 degrees C. Lipids were extracted from lung lavage and lamellar body fractions, and phospholipids and cholesterol components were measured. There was no change in either the total amount or relative proportions of the different classes of phospholipids, but cooling increased the cholesterol content of lavage. An increase in the cholesterol: phospholipid ratio was evident within 2 h of cooling to 19 degrees C and was maintained for at least 48 h. The ratio increased from 8% at 37 degrees C, to 15% after 4 h at 19 degrees C, and 18% after 4 h at 14 degrees C. Possibly the increase in cholesterol promotes fluidity and absorption of surfactant within the alveoli of lizards with low Tb. Cold lizards collapse their lungs during prolonged periods of apnea and the surfactant may prevent the epithelial walls from adhering.

No effect of environmental acidity on the ether glycerophospholipids of crayfish gills

1. Gills of the freshwater crayfish Procambarus clarkii were rich in ether glycerophospholipids (about 35% of total phospholipids). 1'-Alkenyl ethers were more abundant than alkyl ethers in the gill phospholipids. Phosphatidyl ethanolamine was the principal plasmalogen. 2. There were no significant changes in the proportions of the different classes of phospholipids (diacyl, alkyl or alkenyl forms) in the gill lipids of animals acclimated in the laboratory to low pH (4.0–4.5). Although gill membranes are rich in plasmalogens they are relatively stable in a low pH environment. 3. No significant changes in the carbon chains of the ether moieties of alkylglycerols or 1'-alkenylglycerols were detected in gills exposed in vivo to low pH.

30] Modification of phospholipid environment in sarcoplasmic reticulum using nonspecific phospholipid transfer protein

Publisher Summary This chapter focuses on the modification of phospholipid environment in sarcoplasmic reticulum using nonspecific phospholipid transfer protein. The calcium-pump membrane of sarcoplasmic reticulum (SR) from fast twitch skeletal muscle is highly specialized for calcium transport. The calcium-pump protein accounts for 90% of the protein of the calcium-pump membrane of highly purified light SR, which derives from the longitudinal tubules of SR. Phospholipid comprises 35% of the mass of the membrane of which more than 90% is phospholipid. The Ca 2+ -pump protein is oriented transmembrane with a major portion extending out from the cytoplasmic face. The membrane lipids appear to be required for both hydrolysis of ATP and Ca 2+ -pumping activity. The phospholipid exchange procedure consists of incubating the SR membrane vesicles with the transfer protein in the presence of phospholipid vesicles of defined phospholipid composition. The exchange of phospholipid takes place with retention of the unidirectional orientation of the calcium-pump protein in the membrane. The use of a nonspecific phospholipid transfer protein is an effective and gentle way to modify the phospholipid composition in the SR membrane. With this methodology, the presence of two different classes of phospholipids in the SR membranes is observed and interpreted in terms of membrane sidedness.