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PubMed1991

Autoantibodies against all the phospholipids: a comparative systematic study with systemic lupus erythematosus and healthy sera.

The sera of systemic lupus erythematosus patients were tested by ELISA for the presence of autoantibodies against all the phospholipids: cardiolipin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine. The quantity of phospholipid coated (in comparison with that initially deposited) on the microtitration plates was precisely evaluated in order to determine if the results (absorbance values) obtained for each phospholipid could be compared directly. The systemic lupus erythematosus sera tested gave positive results for all the phospholipids. The highest level of autoantibodies was observed with phosphatidic acid followed by phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The sera seemed to contain antibodies directed either against all the 8 phospholipids tested or more specifically against one or two phospholipids. The results were compared with those obtained with 17 healthy sera. Much lower values were obtained for the sera of healthy subjects, the majority of which showed a weak binding, similar for all the phospholipids. These results suggest that the anti-phospholipid autoantibodies present in systemic lupus erythematosus sera are significantly higher than those of healthy subjects. It is concluded that in the investigation of anti-phospholipid antibodies, tests should be carried out for all the phospholipids.

Antibody Specificity↗
PubMed2002

Role of electrostatic and hydrophobic interactions in Ca(2+)-dependent phospholipid binding by the C(2)A-domain from synaptotagmin I.

Most C(2)-domains bind to phospholipid bilayers as a function of Ca(2+). Although phospholipid binding is central for the normal functions of C(2)-domain proteins, the precise mechanism of phospholipid binding is unclear. One of the key questions is whether phospholipid binding by C(2)-domains is primarily governed by electrostatic or hydrophobic interactions. We have now examined this question for the C(2)A-domain of synaptotagmin I, a membrane protein of secretory vesicles with an essential function in Ca(2+)-triggered exocytosis. Our results confirm previous data showing that Ca(2+)-dependent phospholipid binding by the synaptotagmin C(2)A-domain is exquisitely sensitive to ionic strength, suggesting an essential role for electrostatic interactions. However, we find that hydrophobic interactions mediated by exposed residues in the Ca(2+)-binding loops of the C(2)A-domain, in particular methionine 173, are also essential for tight phospholipid binding. Furthermore, we demonstrate that the apparent Ca(2+) affinity of the C(2)A-domain is determined not only by electrostatic interactions as shown previously, but also by hydrophobic interactions. Together these data indicate that phospholipid binding by the C(2)A-domain, although triggered by an electrostatic Ca(2+)-dependent switch, is stabilized by a hydrophobic mechanism. As a result, Ca(2+)-dependent phospholipid binding proceeds by a multimodal mechanism that mirrors the amphipathic nature of the phospholipid bilayer. The complex phospholipid binding mode of synaptotagmins may be important for its role in regulated exocytosis of secretory granules and synaptic vesicles.

Amino Acid Sequence↗
PubMed1990

P-31 NMR analysis of phospholipids from cultured human corneal epithelial, fibroblast and endothelial cells.

Corneal epithelial, fibroblast and endothelial cells, cultured from human donors, were analyzed to determine their characteristic phospholipid profiles by 31P NMR. Tissue phospholipid profiles from epithelial, fibroblast and endothelial cell cultures were evaluated to differentiate the individual cell types and to identify resonances that typically appear in high-resolution phospholipid profiles of whole corneas. Phosphatidylcholine, phosphatidylethanolamine plasmalogen, an uncharacterized phospholipid at 0.13 delta, phosphatidylinositol, phosphatidylserine and sphingomyelin were determined to be, in decreasing order of concentration, the major phospholipids detected in these three cultured corneal cell types. Indices of phospholipid metabolism representing total plasmalogen content, total choline-containing lipids and the total choline-containing lipids less those synthesized through the plasmalogen pathway were found to differentiate the three cell types. Minor phospholipids cardiolipin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine (LPC) and LPC plasmalogen not usually reported in studies of corneal phospholipids using other techniques, were useful in discriminating between cell types. Phospholipid profiles of the whole cornea provide important information concerning the biochemistry and pathology of the tissue, however, phospholipid analysis of individual components of the cornea, such as the epithelial, fibroblast and endothelial cells, makes it possible to understand the contribution of specific cellular constituents to the spectral information obtained from the whole cornea.

Cells, Cultured↗
PubMed1990

Effect of verapamil on CRF-induced abnormalities in phospholipid contents of brain synaptosomes.

Chronic renal failure is associated with significant reductions in total phospholipids, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine of brain synaptosomes. These derangements in synaptosomal phospholipid metabolism were attributed to the state of secondary hyperparathyroidism of chronic renal failure (CRF) and the parathyroid hormone-induced accumulation of calcium in synaptosomes. This study examined whether a calcium channel blocker, verapamil, would prevent this synaptosomal calcium accumulation and correct the abnormalities in synaptosomal phospholipids in CRF. Verapamil treatment of normal rats for 21 days did not affect synaptosomal content of calcium or phospholipids. CRF of 21 days' duration was associated with a significant (P less than 0.01) increase in synaptosomal calcium (10.2 +/- 0.5 vs 7.4 +/- 0.6 nmol/mg protein) and a significant reduction (P less than 0.01) in total phospholipids (397 +/- 12 vs 529 +/- 19 nmol phospholipid P/mg protein), phosphatidylinositol (2.7 +/- 0.22 vs 4.6 +/- 0.27 nmol phospholipid P/mg protein), and phosphatidylserine (37 +/- 1.9 vs 83 +/- 5.2 nmol phospholipid P/mg protein). Simultaneous treatment of CRF rats with verapamil for 21 days reversed the synaptosomal abnormalities in calcium and phospholipid contents. Our data support the notion that the effect of excess parathyroid hormone of CRF on synaptosomal phospholipids is mainly due to the parathyroid hormone-induced calcium accumulation.

Animals↗
PubMed2001

Soluble CD14 mediates efflux of phospholipids from cells.

Soluble CD14 (sCD14), a 55-kDa glycoprotein found in plasma, has been shown to act as a shuttle for bacterial LPS and phospholipids, transporting LPS and phospholipid monomers from LPS aggregates or liposomes to high density lipoprotein particles. sCD14 has also been shown to mediate the transport of LPS and phosphatidylinositol into cells. Here we show that sCD14 mediates not only the influx but also the efflux of cellular phospholipids. Addition of sCD14 enhanced efflux of cellular phospholipids labeled with [(3)H]palmitic acid, [(3)H]oleic acid, or [(3)H]choline chloride from differentiated THP-1 monocytic cells. Efflux was dependent on the concentration of sCD14 added and was essentially complete in 30 min. The role of membrane-bound CD14 (mCD14) in lipid efflux was assessed using matched pairs of cell lines that express or fail to express this protein. While efflux was very dependent on mCD14 in U373 cells, it was not dependent on mCD14 in Chinese hamster ovary cells, suggesting a role for additional cellular proteins in determining the pathway of phospholipid efflux. A deletion mutant of sCD14 lacking the LPS binding site had less ability to efflux phospholipids than intact sCD14, suggesting that this site is needed for CD14 to serve in phospholipid transport. [(3)H]Palmitate-labeled lipids released by sCD14 were precipitated with anti-CD14 then analyzed by HPLC. Phosphatidylcholine was the dominant phospholipid exported and bound to sCD14. These results demonstrate that sCD14 mediates efflux of phospholipids from cells and suggest that sCD14 contributes to phospholipid transport in blood.

Acute-Phase Proteins↗
PubMed2000

Phospholipids in dialysate and the peritoneal surface layer.

Dialysate concentration of phospholipids has been used to monitor peritoneal membrane status. However, we recently found that the peritoneum has a surface layer in which phospholipids may be the main constituent. Therefore, in this study, we compared the phospholipids composition of peritoneal dialysate and of the peritoneal surface in rats. Eight male Sprague-Dawley rats were used in the study. Five rats received an intraperitoneal injection of 25 mL 4.25% glucose dialysis solution. After four hours, the rats were killed, and the dialysate was drained completely. Then 20 mL of Folch solution was infused into the peritoneal cavity for 30 seconds and drained completely. The other three rats received the Folch solution without dialysis. The effluent and Folch solution were then processed for phospholipids analysis using high performance thin-layer chromatography (HPTLC). The total phospholipids content was ten times higher in the surface layer than in the dialysate effluent. In the effluent, four clearly different components were seen: lysophosphatidylcholine (LPC), sphingomyelin (SM, 29%), phosphatidylcholine (PC, 66%), and phosphatidylinositol (PI, 4.5%). However, in the surface layer, as well as LPC, SM (20.6%), PHC (47%), and PI (6.3%), two additional components were seen, phosphatidylserine (PS, 17.1%) and phosphatidylethanolamine (PE, 8.9%). The quantity of phospholipids in the peritoneal surface of non dialyzed rats was similar to the total quantity of phospholipids (in effluent and in the peritoneal surface) of dialyzed rats. Our results suggest that: (1) a surface layer is present on the peritoneum; this layer could well be extracted by Folch solution; and, with appropriate incubation time, one can separate the surface layer without damaging the mesothelial cells; (2) the composition of phospholipids in the effluent is different from that in the peritoneal surface layer, which contains membrane phospholipids (PS and PE); (3) shielding from the peritoneal surface may be the main reason for the presence of phospholipids in the dialysate.

Animals↗
PubMed2001

The influence of lipid composition and divalent cations on annexin V binding to phospholipid mixtures.

Annexin V is a 36-kDa protein which, it has been suggested, is a factor in protecting the vascular endothelium from attack by antibodies to other phospholipid-binding proteins. Competition between annexin V and beta2-glycoprotein I (beta2GPI) for phospholipid surfaces is complicated by empirical observations regarding alterations in binding to anionic phospholipid, primarily phosphatidylserine. In order to elucidate the effect of phospholipid composition and divalent cations (Ca(+2) and Mg(+2)) on annexin V binding to phospholipid, we used biotinylated annexin V and peroxidase-conjugated avidin D to probe the binding of annexin V to phospholipid-coated wells of polystyrene microtiter plates. Binding of annexin V to anionic phospholipid is Ca(+2)-dependent and, in its absence, annexin V was found to bind most avidly to 100% phosphatidylcholine in a saturable manner, followed by decreasing percentages of phosphatidylcholine. Ca(+2) was found to inhibit phosphatidylcholine binding and promote the binding of phospholipid mixtures containing phosphatidylserine. Phosphatidylserine (100%) did not bind annexin V as strongly as mixtures of 50% and 75% phosphatidylserine. The effect with Ca(+2) suggests saturation of Ca(+2)-binding sites on annexin V, reached under our experimental conditions at approximately 1 mM. Under the same conditions, Mg(+2) slightly enhanced the binding of all of the phospholipid compositions studied. Ca(+2)-dependent binding of annexin V was competitively inhibited by Mg(+2); 5 mM Mg(+2) reduced binding significantly (p < 0.0001 by ANOVA, p < 0.05 for post hoc test of 5 mM vs 0 mM). These data suggest that the translocation of membrane phospholipid under the dynamics of ion transport in vascular endothelium may alter annexin V binding.

Annexin A5↗
PubMed2005

[Preparation of silybin-phospholipid complex and its bioavailability in rats].

AIM: To prepare silybin-phospholipid complex and study its physicochemical properties. To compare the pharmacokinetic characteristics and bioavailability after oral administration of silybinphospholipid complex and silybin material in rats. METHODS: Using acetone as a reaction medium, silybin and phospholipid were resolved into the medium, when the organic solvent was clear, then removed under vacuum evaporation, silybin-phospholipid complex was obtained. The new complex' s physicochemical properties including DSC, UV, IR were determined. The concentrations of non-conjugated and total silybin after oral administration of silybin-phospholipid complex and silybin material at different time in rats were determined by RP-HPLC. The pharmacokinetic parameters were computed by software program 3P97. RESULTS: Experiment results showed that silybin and phospholipid in the silybin-phospholipid complex were combined by non-covalent-bond, not forming a new compound and the solubility of silybin-phospholipid complex in water and n-octanol was effectively enhanced. It was found that mean plasma concentration-time curve of silybin after oral administration of silybin-phospholipid complex in rats was in accordance with one-compartment model with first-order absorption. Pharmacokinetic parameters of non-conjugated and total silybin in rats were respectively T(max) 10 min and 2 h; C(max) 0.11 and 1.08 microg x mL(-1); T1/2 2.18 and 3.84 h; AUC(0-infinity) 1.71 and 12.94 microg x mL(-1) x h. However, after oral administration of silybin material, plasma levels of both non-conjugated and total silybin were within the analytical detection limit. CONCLUSION: It was concluded that after oral administration of silybin-phospholipid complex in rats the bioavailability of silybin increased greatly. This was mainly due to an obvious improvement of the lipophilic property of silybin-phospholipid complex compared with silybin material and an increase in gastrointestinal absorption.

Administration, Oral↗
PubMed1990

[Effect of mercuric chloride on phospholipid peroxidation in rat].

Individual molecular species of mercuric chloride induced phospholipid peroxide formed in rat brain, liver and kidney were determined using the multi-channel UV-high-performance liquid chromatography (HPLC) combined with potentiometric determination. Mercuric chloride (0.5 mg/kg/day) was administered subcutaneously to rats for 3 days. In accordance with a specified time schedule following administration (0.5, 1, 3 and 5 days), rats were decapitated and the phospholipids of brains, livers and kidneys were extracted and purified. The samples were then injected into the HPLC and the ratio (235 nm/203 nm) of peak area of each phospholipid was calculated. The peroxide value of each sample was determined using the calibration curve of the auto-oxidized standard phospholipids which were analyzed by both potentiometric determination and UV HPLC. Kidney phospholipid peroxides were easier induced than those in other organs reached their maximum peak (20.5 meq./kg) at 1 day after initial administration. Phospholipid peroxides in kidney and brain showed similar movement, while those in liver showed their maximum peak 3 days later. Of the phospholipids, phosphatidylserine and phosphatidylethanolamine seemed to be more susceptible to lipid peroxidation induced by mercuric chloride, the common feature of these two phospholipids being that both of them have primary amine group(s) on their polary heads and both are located on the cytosolic side of cell membrane. These results may be explained by mechanisms that relate to the interaction between mercurials and cell membranes or that between mercurials and primary amine groups of phospholipids. Further study is necessary to clarify the specific mechanisms involved in the induction of lipid peroxidation by mercurials and the interaction of mercurials with phospholipids.

Animals↗
PubMed1979

Role of phospholipids in the structure and function of the thyrotropin receptor.

Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.

Animals↗
PubMed1985

Retrograde axonal transport of endogenous phospholipids in rat sciatic nerve.

Anterograde axonal transport of phospholipids occurs at a rate of several hundred millimeters per day. However, although labeled precursors are incorporated into phospholipids in the neuronal cell bodies within several hours, these newly synthesized phospholipids are committed to transport over a much longer period of time. Thus, maximal accumulation of radioactive lipids in axons and nerve endings does not occur for several days (e.g., 4 to 7 days in rat optic tract and sciatic nerve). We have now investigated the retrograde axonal transport of endogenous phospholipid molecules in sensory neurons of rat sciatic nerve. Labeled phospholipids were delivered to axons and nerve endings of these cells by anterograde axonal transport following injection of [2-3H] glycerol into the L5 dorsal root ganglion. At various times following precursor injection two ligatures, 9 mm apart, were applied to the mid-thigh region of the sciatic nerve. Animals were sacrificed 3 to 48 hr after nerve ligation, nerves were dissected and sectioned into 5-mm segments, and phospholipid radioactivity in each segment was determined. The time-dependent accumulation of labeled phospholipids distal to the distal ligature demonstrated their retrograde axonal transport. The time course of retrograde transport for these phospholipids was more prolonged and peaked several days later than the time course for the anterograde transport phase. Further information regarding the relationship between radioactive phospholipids arriving at the nerve endings by anterograde transport, and their subsequent "turn-around" and retrograde transport back to the nerve cell bodies, was obtained by analyzing the phospholipid class label distribution of both of these transport phases at various times following precursor injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

The transport and turnover of phospholipids in the rat nigrostriatal system: effects of d-amphetamine and haloperidol.

the nigrostriatal transport of phospholipids was studied using [3H] glycerol, 32Pi and [3H] choline. [3H] glycerol was rapidly incorporated into phospholipid and significant amounts of labelled phospholipid were found in the striatum one hour after injection into the substantia nigra. In contrast, both 32Pi and [3H] choline were more slowly incorporated into phospholipid and significant amounts of labelled phospholipid were not found in the striatum until 24 hours after injection. Once incorporated into the striatum, the [3H-glycerol] phospholipids showed both a rapid (t 1/2 = 1-4 days) and a slow (t 1/2 = 14 + days) turnover component while the [32p] and [3H-Ch] phospholipids showed only a slow turnover component. The subcellular distribution of the rapidly transported [3H-glycerol] phospholipids was studied. Only [3H] phosphatidylcholine (PC) was specifically enriched in the synaptic membrane fraction. The hypothesis was tested that an increase in vesicular lysophosphatidylcholine (LPC) content is associated with dopamine (DA) release. The DA containing vesicles in the striatum were labelled by the intranigral injection of [3H]choline; seven days later, the animals were administered haloperidol to stimulate firing of the nigral-striatal DA neurons. Haloperidol significantly decreased rather than increased the [3H]LPC/[3H]PC ratio. The hypothesis was tested that chronic amphetamine treatment would inhibit phospholipid transport as a result of the decrease in neuronal activity. Chronic d-amphetamine treatment was found to have no effect on the fast component of [3H-glycerol] phospholipid turnover.

Animals↗
PubMed1983

Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles.

Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.

Animals↗
PubMed1983

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids.

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.

Animals↗
PubMed1981

Vesicle-mediated transfer of phospholipids to plasma membrane during cell aggregation of Dictyostelium discoideum.

We have previously reported that synthesis of phospholipids increases 4-fold at the onset of chemotactic migration during development of Dictyostelium discoideum and that the newly synthesized phospholipids are preferentially incorporated into the plasma membrane (De Silva, N. S. and Siu, C-H. (1980) J. Biol. Chem. 255, 8489-8496). To test the hypothesis that the rapid transfer of phospholipids to the plasma membrane is mediated by vesicles, we isolated phospholipid-rich vesicles from cells at 6 h of development. These vesicles had an average size of 0.35 micrometer in diameter. They banded at a density of 1.097 g/cm3 and they had a phospholipid: protein (w/w) ratio of 2.25. The predominant classes of phospholipids in these vesicles were phosphatidylethanolamine and phosphatidylcholine. In pulse-labeling studies using [3H]glycerol, these low density vesicles had the highest phospholipid-specific activity, which was about 3 times higher than that of 6-h plasma membranes. Almost 80% of the incorporated radioactivity was found to be associated with phosphatidylethanolamine and phosphatidylcholine. When cells were chased with cold precursor after pulse labeling, the specific activity of these vesicles dropped by almost 20-fold in 90 min, while plasma membranes showed a 2.5-fold increase in 60 min. Addition of colchicine to 7-h cells inhibited the translocation of newly synthesized phospholipids to the plasma membrane. The low density vesicles were found in much reduced amounts in preaggregation stage cells or the aggregateless mutant WL3. These results indicate that transfer of newly synthesized phospholipids from their site of synthesis to the plasma membrane probably occurs through a special class of phospholipid-rich vesicles.

Cell Fractionation↗
PubMed1980

Investigation of the inside-outside distribution, intermembrane exchange and transbilayer movement of phospholipids in sonicated vesicles by shift reagent NMR.

1. A new NMR approach is described for the investigation of transbilayer asymmetry in phospholipid vesicles consisting of phosphatidylcholine and negatively charged phospholipids. The method makes use of the dependence of the psuedocontact shift of the N-methyl proton resonance induced by paramagnetic ions on the surface concentration of negatively charged phospholipids. When two differently shifting paramagnetic probes are applied from the outside and the inside of a vesicular membrane the transbilayer phospholipid distribution can be estimated without knowledge of the inner and outer radii of the vesicles and the packing density of the phospholipid molecules. 2. The method was employed to study the transbilayer asymmetry in vesicles obtained by cosonication of phosphatidylcholine with phosphatidylserine, phosphatidylglycerol or phosphatidylinositol. The three negative phospholipids were found to distribute with a higher surface concentration in the inner vesicular shell than in the outer one when their total content did not exceed 25 mol%. However, as the amount of negatively charged phospholipids increases the ratio of their inside to outside surface concentrations, i.e., the transbilayer asymmetry of the vesicles, decreases. Prolonged incubation (for several days) does not change the compositional asymmetry of the cosonicated vesicles. 3. By the 'double-probe' technique it was established that spontaneous exchange between separately sonicated phosphatidylcholine and phosphatidylinositol vesicles results in formation of highly asymmetric mixed vesicles with phosphatidylinositol residing only in the outer monolayer. In the presence of antioxidant (alpha-tocopherol) the bilayer asymmetry is preserved for days. However lipid peroxidation induces rapid transbilayer movement (flip-flop) of phospholipids leading to an 'inverted' asymmetry resembling that of cosonicated vesicles. It is suggested that lipid peroxidation promotes phospholipid flip-flop by partially converting the bilayer structure into a non-bilayer configuration. Moderate quantities of lysophosphatidylcholine (up to 15 mol%) induce neither detectable perturbations of the bilayer nor rapid phospholipid flip-flop.

Electrochemistry↗
PubMed1995

Quantitative chromatographic analysis of inositol phospholipids and related compounds.

The metabolism of phospholipids and the mobilization of second messengers such as inositol-1,4,5-trisphosphate, 1,2-diacylglycerol (DAG) and arachidonic acid (AA) from phospholipids is commonly studied by radiolabelling phospholipids with [3H]myo-inositol or [32P]ATP and measuring the incorporation of radioactivity in different phospholipids or their hydrolysis products. However, for the radiolabelling method to accurately reflect changes in the compound's mass, it is essential that the tissue is labelled to isotopic equilibrium which is difficult to achieve. To circumvent the disadvantages of the radiolabelling method, several analytical procedures have been developed for the mass analysis of phospholipids and inositolphosphates (IPs). Quantitation of the mass or the radiolabelling of phospholipids is a complex multi-step procedure that involves quantitative isolation of phospholipids, fractionation of individual phospholipids and either determination of radioactivity in each component or the measurement of their mass. Phospholipids, DAG and AA are extracted from tissue sample with organic solvents such as chloroform-methanol (2:1) containing HCl or formic acid. The extract is separated by TLC, cartridge-column chromatography or HPLC on a reversed-phase column. Phospholipids are quantitated by measuring inorganic phosphate, absorption at 200 nm or mass spectrometry. Inositol phosphates are extracted with perchloric acid or trichloroacetic acid and separated by ion-exchange cartridge-column or HPLC with an ion-exchange column. IPs are quantitated by measuring inorganic phosphate or by using enzymatic reaction, metal-dye coupling, NMR or mass spectrometry.

Arachidonic Acid↗
PubMed1996

Antigen-induced generation of lyso-phospholipids in human airways.

The goal of the current study was to examine the formation of phospholipids, 1-radyl-2-lysosn-glycero-phospholipids (lyso-PL) and 2-acetylated phospholipids (such as PAF) as well as mechanisms responsible for generating these phospholipids in bronchoalveolar lavage fluid (BAI.F) from allergic subjects challenged with antigen. Bronchoalveolar lavage was performed in normal and allergic subjects before, 5-30 min, 6 h, and 20 h after segmental antigen challenge via a wedged bronchoscope. Levels of 1-hexadecyl-2-lyso-phospholipids and 1-hexadecyl-2-acetyl-phospholipids were initially determined by negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS). Antigen dramatically elevated quantities of 1-hexadecyl-2-lyso-phospholipids in allergic subjects 20 h after challenge when compared to non-allergic controls. In contrast, there was not a significant increase in levels of 1-hexadecyl-2-acetyl-phospholipids after antigen challenge. Closer examination of 1-radyl-2-lyso-sn-glycero-3-phosphocholine (GPC) revealed that 1-palmitoyl-2-lyso-GPC, 1-myristoyl-2-lyso-GPC and 1-hexadecyl-2-lyso-GPC were three major molecular species produced after antigen challenge. 1-palmitoyl-2-lyso-GPC increased sevenfold to levels of 222 +/- 75 ng/ml of BALF 20 h after antigen challenge. The elevated levels of lyso-PL correlated with levels of albumin used to assess plasma exudation induced by allergen challenge. In contrast, the time course of prostaglandin D2 (PGD2) or 9 alpha, 11 beta PGF2 (11 beta PGF2) formation did not correlate with lyso-PL generation. To examine the mechanism leading to lyso-phospholipid formation in antigen-challenged allergic subjects, secretory phospholipase A2 (PI.A2) and acetyl hydrolase activities were measured. There was a significant increase in PLA2 activity found in BALF of allergic subjects challenged with antigen when compared to saline controls. This activity was neutralized by an antibody directed against low molecular mass, (14 kD) human synovial PLA2 and dithiothreitol. Acetyl hydrolase activity also markedly increased in BALF obtained after antigen challenge. This study indicates that high levels of lyso-PLs are present in airways of allergic subjects challenged with antigen and provides evidence for two distinct mechanisms that could induce lyso-PL formation. Future studies will be necessary to determine the ramifications of these high levels of lyso-phospholipids on airway function.

Adult↗