Distributions of hydroperoxide positional isomers generated by oxidation of 1-palmitoyl-2-arachidonoyl-<Emphasis Type="Italic">sn</Emphasis>-glycero-3-phosphocholine in liposomes and in methanol solution

The differences in distribution of geometric isomers of unsaturated PC hydroperoxides generated by free radical oxidation were compared, as corresponding hydroxy analogs, in heterogeneous liposomes and in a homogeneous methanol solution by using HPLC with UV detection due to the presence of conjugated dienes. Identification of fractionated peak components was carried out by GC-MS. When the oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, PC(16∶0/18∶2), was initiated in liposomes by a hydrophilic azo radical initiator, and in a methanol solution by a hydrophobic azo radical initiator, there was no significant difference in the relative percentages of 1-palmitoyl-2-(9-hydroxy-trans-10,trans-12-octadecadienoyl)-sn-glycero-3-phosphocholine (9-t,t-OH PC) and 1-palmitoyl-2-(13-hydroxy-trans-9,trans-11-octadecadienoyl)-sn-glycero-3-phosphocholine (13-t,t-OH PC) between the PC oxidized in liposomes and in the methanol solution. For the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, PC(16∶0/20∶4), the relative percentage of 1-palmitoyl-2-(5-hydroxy-trans-6,cis-8,11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (5-OH PC) was significantly higher (P<0.01) than that of 1-palmitoyl-2-(15-hydroxy-cis-5,8,11,trans-13-eicosatetraenoyl)-sn-glycero-3-phosphocholine (15-OH PC) in liposomes. For the homogeneous methanol solution of PC(16∶0/20∶4), the relative percentage of 5-OH PC was close to that of 15-OH PC. For the PC(16∶0/20∶4) oxidized in bulk with added pentamethylchromanol, the individual amount of 15-OH PC, 1-palmitoyl-2-(11-hydroxy-cis-5,8trans-12,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (11-OH PC), 1-palmitoyl-2-(12-hydroxy-cis-5,8,trans-10,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (12-OH PC), 1-palmitoyl-2-(8-hydroxy-cis-5,trans-9,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (8-OH PC), 1-palmitoyl-2-(9-hydroxy-cis-5,trans-7,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (9-OH PC), and 5-OH PC were close to each other compared to the corresponding values in liposomes and in methanol solution. The results obtained by gel permeation chromatography of the PC liposomes containing hydrophilic 2,2′-azobis-2-amidinopropane) dihydrochloride (AAPH) suggest that the AAPH added to the liposomes of PC(16∶0/20∶4) was partitioned into the water phase and out of the hydrophobic region of the fatty acyl moieties of the PC. These results confirm that the distance that exists in the bis-allylic carbons of the unsaturated fatty acyl moieties of PC from the interface between the hydrophilic region of PC and the water phases played an important role in influencing hydrogen abstraction to form a symmetrical distribution of hydroperoxide isomers in both the heterogeneous liposomes and the homogeneous methanol solution.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.     

Molecular Dynamics and Partitioning of Di-<Emphasis Type="Italic">tert</Emphasis>-butyl Nitroxide in Stratum Corneum Membranes: Effect of Terpenes

In this work, we have used electron paramagnetic resonance (EPR) spectroscopy of the small spin label di-tert-butyl nitroxide (DTBN), which partitions the aqueous and hydrocarbon phases, to study the interaction of the terpenes α-terpineol, 1,8-cineole, l(−)-carvone and (+)-limonene with the uppermost skin layer, the stratum corneum, and the membrane models of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The EPR spectra indicated that the terpenes increase both the partition coefficient and the rotational correlation time of the spin labels in the stratum corneum membranes, whereas similar effects were observed in the DMPC and DPPC bilayers only at temperatures below the liquid-crystalline phase. The EPR parameter associated to probe polarity inside the membranes showed thermotropically induced changes, suggesting relocations of spin probe, which were dependent on the membrane phases. While the DMPC and DPPC bilayers showed abrupt changes in the partitioning and rotational correlation time parameters in the phase transitions, the SC membranes were characterized by slight changes in the total range of measured temperatures, presenting the greatest changes or membranes reorganizations in the temperature range of ~50 to ~74 °C. The results suggest that terpenes act as spacers, weakening the hydrogen-bonded network at the polar interface and thus fluidizing the stratum corneum lipids.     

Molecular species composition of phosphatidylcholines during the development of the avian embryo brain

A comparative approach has been used to investigate the molecular species composition of phosphatidylcholine (PC) and its age variation throughout several developmental stages of chick and duck embryo brains. The brain PC consist of 15 major molecular species which do not undergo appreciable variation in their relative abundance either during embryonic development or between equivalent stages of maturation in the 2 avian species. In fact, a highly invariable molecular architecture of PC is shown in the developing organ. Molecular species containing saturated or monounsaturated fatty acids were dominant in all stages of development of the avian embryo brain. Among these molecular species, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine accounted for 75–80% of the total PC.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

Intestinal absorption of ester and ether glycerophospholipids in guinea pig. Role of a phospholipase A2 from brush border membrane

In vivo intestinal perfusion was used to follow the absorption of three different choline glycerophospholipids (CGP) in guinea pig. These included 1-[³H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine (diacyl-GPC), 1-[³H]-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phospho-[³H]-choline (dialkyl-GPC). About 80% of diacyl-GPC was absorbed within 4 hr, compared to 60% of alkylacyl-GPC and 30% of dialkyl-GPC. The radioactivity disappearing from the perfusion fluid was recovered in intestinal lipids, mostly triacylglycerol, free fatty acid and CGP from diacyl-GPC, CGP from alkylacyl-GPC and dialkyl-GPC. These results indicated that the nonhydrolyzable substrate dialkyl-GPC was much less absorbed, whereas diacyl-GPC, which released over 80% of [³H]palmitic acid in the perfusion fluid, displayed the highest absorption rate. The intermediate picture observed for alkylacyl-GPC suggested the possible involvement of a phospholipase A₂, which was detected in the entire intestinal tract. This enzyme was further found to concentrate in villus cells, where it is localized in the brush border membrane, as shown using two different subcellular fractionation procedures. These data suggest a possible role of this new enzyme in the digestion of alimentary phospholipids.     

Solubilization of phospholipid vesicular structures into mixed micelles of zwitterionic surfactants

Interactions between the binary combination of dimethyltetradecylammoniopropanesulfonate (TPS) and l-α-phosphatidylcholine (PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the aqueous bulk phase were evaluated with the help of pyrene fluorescence (l ₁/l ₃) measurements by studying the aggregation processes of TPS in pure water and in the presence of 7–36 μM of fixed concentrations of each lipid. The fluorescence measurements showed that TPS monomers undergo two kinds of aggregation process, which were identified by the three breaks. The first break, C₁, and the second, C₂, indicated the onset and completion of bilayer solubilization, respectively, on the incorporation of TPS monomers into the bilayer assemblies, which led to bilayer solubilization in the form of mixed micelles. This process was not clearly visible in the presence of PC, whereas some kinds of structure transitions were observed upon the incorporation of surfactant monomers. The partition coefficient (K), which defines the degree of partitioning of surfactant monomers into the bilayers with respect to the aqueous medium, was evaluated. A high K value of TPS-lipid aggregates indicated stronger interactions between surfactant and bilayer assemblies of lipid. The K values determined for the three phospholipids are close to each other, which indicates that K values do not depend on the hydrocarbon chain length of the phospholipid but of the surfactant used.     

Inhibiting the vascular smooth muscle cells proliferation by EPC and DPPC liposomes encapsulated magnolol

Percutaneous transluminal coronary angioplasty (PTCA) is a non-surgical modality for treating stennosis. However, the recurrence of restenosis in 30-50% patients within 6 months is the major drawback of PTCA. The major reason of restenosis is the proliferation of the vascular smooth muscle cells (VSMCs). Magnolol, a pure compound extracted from Magnolia officinalis, encapsulated by liposome was investigated for inhibiting the VSMCs proliferation leading to restenosis by PTCA. 1,2-Diacyl-Sn-glycero-3-phosphocholine (EPC) and 1,2-dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC) liposomes were utilized to encapsulate the magnolol. EPC liposome obtained the higher encapsulation efficiency than DPPC lipsomes from UV-vis spectroscopy study. The inhibiting efficiency of EPC and DPPC liposomes encapsulated magnolol was higher than pure magnonol. Magnolol encapsulated by EPC liposomes had better efficiency on inhibiting VSMCs than DPPC liposome. Addition of cholesterol in liposomes could slightly enhance the encapsulation efficiency. The particles sizer analysis revealed the average particles size of EPC and DPPC liposomes encapsulated magnolol became larger than pure EPC or DPPC liposomes. From the transmission electron microscopy (TEM) analysis, the magnolol seems to interfere with EPC and DPPC liposomes to form a homogeneous lipid bilayer.     

Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Leukotriene B4 promotes phospholipid acylation in human neutrophils

The present study was undertaken to test the hypothesis that leukotriene B₄ (LTB₄) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB₄ (1.5 to 150 nM) for 1 for 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferases catalyzing acylation of the respective lysophospholipids. LTB₄ stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid but not by arachidonic acid. LTB₄ and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB₄ or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB₄-activated neutrophils reflects activation of phospholipase A₂ and acyltransferases as well as ofde novo phospholipid synthesis.     

Purification of phospholipids by preparative high pressure liquid chromatography

A method is described for the purification of a number of phospholipids by preparative high performance liquid chromatography (HPLC). Purification of digalactosyl-diglyceride from spinach and egg phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine from its reaction mixture have been resolved. The lipid separation is performed on a polygosil column and the individual compounds are monitored directly by refractive index detection. Chloroform/methanol mixtures are used as eluent systems, providing a wide polarity range to separate the classes of lipids. The developed equipment can be used for columns between 10 and 50 cm long and 4 and 50 mm inner diameter. The flow rate could be varied between 1 and 100 ml/min and applied pressures between 10 and 450 bars.     

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [³²P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine to the medium,¹⁴C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,¹⁴C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the¹⁴C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min. Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A₁ and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A₂ was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A₁ and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.     

Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase D-catalyzed transphosphatidylation

A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested. The reaction proceeded well, giving almost no hydrolysis of PtdCho to PtdOH in a biphasic system consisting of diethyl ether and acetate buffer at 30°C. The isopropylidene protective group was removed by heating the diastereomeric isopropylidene PtdGro at 100°C in trimethyl borate in the presence of boric acid to obtain the desired PtdGro diastereomers. The purities of the products, which were determined by chiral-phase HPLC, were exclusively dependent on the optical purities of the original isopropylideneglycerols used. The present method is simple and can be utilized for the synthesis of pure PtdGro diastereomers having saturated and unsaturated acyl chains.     

Aggregated assemblies of hexadecyltrimethylammonium bromide and phospholipids at the interface and in the bulk solution

Interactions between hexadecyltrimethylammonium bromide (HTAB) and l-α-phosphatidylcholine (PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PPC) at the air/water interface and in the bulk solution were evaluated using interfacial tension, fluorescence, and conductivity measurements to study the aggregation processes of HTAB in fixed concentrations of aqueous lipid solutions containing 7–36 μM of each lipid. The interfacial and fluorescence measurements showed the occurrence of two kinds of aggregation processes in HTAB-PC and HTAB-DPC systems, which were identified by the three breaks at low lipid concentrations, i.e., 7–14μM. The first aggregation process, C ₁, involved the incorporation of HTAB monomers into the lipid vesicles and the subsequent disruption of the vesicular structure leading to the formation of mixed micelles. The second aggregation process, C ₂, which started at the second break, indicated the completion of the mixed micelles and the initiation of independent micelle formation process which was completed at the third break. At higher PC or DPC concentration, i.e., 14–22 μM, the first break, which indicated the initiation of the first aggregation process, was not observed; only the second and third breaks were visible. This is ascribed to the presence of comparatively large vesicles, which were able to accommodate HTAB monomers initially without any drastic change in their architecture. In the presence of PPC, the aggregation process of HTAB was similar to that in pure water as studied from the interfacial tension measurements, whereas the bulk behavior indicated the presence of strong structure transitions upon incorporation of HTAB monomers in the PPC vesicles. A difference between the aggregation behaviors of HTAB with PPC was explained on the basis of the stronger hydrophobicity of PPC over PC and DPC.     

Polymerizable glycerophosphocholines containing terminal 2,4-hexadienyloxy groups and their polymerized vesicles(1)

Summary Polymerizable glycerophosphocholines containing one or two 2,4-hexadienyloxy groups at the terminal of the acyl chains were prepared. Those were 1-[11-(2,4-hexadienyloxy)undecanoyl]-2-0-alkyl-rac-glycero-3-phosphocholines 1, 1-acyl-2-[11-(2,4-hexadienyloxy)undecanoyl]-sn-glycero-3-phosphocholines 2 and 1,2-bis[11-(2,4-hexadienyloxy)undecanoyl]-sn-glycero-3-phosphocholine 3. Those having one hexadienyloxy group formed small unilamellar vesicles. One having two groups formed lipid bilayers, but not unilamellar vesicles. 1 and 2 could form stable microcapsules (polymerized vesicles) with the diameters ranging from 20 to 40 nm.     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First,rac-1(3)-O-alkylglycerol is prepared by alkylation ofrac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine givesrac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A₂ (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The “unnatural” 3-O-alkyl-2-O-acyl-sn-glycerol-1-phosphocholine enantiomer, which is not susceptible to phospholipase A₂ cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.     

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat

The importance of the steric configuration of lysophosphatidylcholine in the lymphatic transport of fat was investigated in bile fistula rats. It was found that the feeding of 1-palmitoyl-sn-glycero-3-phosphocholine increased the lymphatic output of phosphatidyl choline and triacylglycerol, while the feeding of 3-palmitoyl-sn-glycero-1-phosphocholine had no effect. In intestinal microsomes of the bile fistula rats, it was found that the lysophosphatidylcholine acyltransferase was stereospecific in acylating the 1-acyl-sn-glycero-3-phosphocholine enantiomer. The significance of these findings is briefly discussed.