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.     

Separation of isomeric lysophospholipids by reverse phase HPLC

A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 orsn-2 position) and the position (Δ⁶ or Δ⁹) or geometric configuration (cis ortrans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at thesn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both thesn-1 andsn-2 isomers of monounsaturated species increased in the order Δ⁹-cis < Δ⁹-trans < Δ⁶-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ⁹-cis and Δ⁶-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.     

1-O-alkyl-linked phosphoglycerides of human platelets: Distribution of arachidonate and other acyl residues in the ether-linked and diacyl species

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l′-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l′-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-l′-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16∶0, 18∶0, (Δ⁹), 18∶2(n−6) and 20∶4(n−6). The 1-O-alk-l and 1-O-alk-l′-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22∶4(n−6), 22∶5(n−3) and 22∶6(n−3) acyl chains. Arachidonate comprised 44% of the acyl residues in thesn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-l′-enyl-2-acyl species were 23% and 25%, respectively, based on all 20∶4(n−6) being linked to thesn-2 position of all classes. In the ethanolamine-linked phosphoglycerides, arachidonate constituted 60%, 20% and 68% of the acyl groups in thesn-2 position of the 1,2-diacyl, 1-O-alkyl-2-acyl and 1-O-alk-l′-enyl-2-acyl classes, respectively. The content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine appears sufficient to support the synthesis of platelet activating factor by a deacylation-reacylation pathway in platelets. Our findings also demonstrate that human platelets contain a significant amount of 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine that could possibly serve as a precursor of both platelet activating factor and bioactive arachidonate metabolites.     

1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonitoEuthynnus pelamis (Linnaeus)

The existence of ether-linked phospholipids, including 1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and ethanolamines in bonitoEuthynnus pelamis (Linnaeus) white muscle, was investigated by gas chromatography and gas chromatography-mass spectrometry. Chemical ionization (iso-butane) mass spectrometry of trimethylsilyl ethers derived from the corresponding ether-linked glycerophospholipids proved effective not only for determining molecular weights but also for structural identification based on the ions [M−R]⁺, [M−RO]⁺ and [M+1]⁺. 1-O-Alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine accounted for 3.0–6.0% and 3.6–7.6% of the total glycerophospholipids, respectively. 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine and ethanolamine were also determined for one fish and accounted for 1.4% and 0.6% of the total glycerophospholipids, respectively. The predominant long chains in thesn-1 position of the glycerol moieties were 16∶0, 18∶0 and 18∶1 in the case of the alkenylacyl and alkylacyl components. Fatty acid distribution of individual glycerophospholipids was also determined.     

Biphasic action of platelet-activating factor on isolated guinea-pig ileum

1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10⁻¹⁰-10⁻⁹ M induced slow contraction of isolated guinea-pig ilcal muscles and the contraction persisted for a long time. At a higher concentration of 10⁻⁷ M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10⁻⁶-10⁻⁵ M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetylcholine-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the dual effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.     

Molecular species analysis of phosphoglycerides from the ripe roes of cod (Gadus morhua)

Molecular species of the 3,5-dinitrobenzoyl derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were quantitated by UV detection at 254 nm after reversed-phase HPLC using solvent systems modified from Takamuraet al. (Lipids 21, 356–361, 1986). Three isocratic solvent systems were used and a total of 39 different molecular species detected. Four species, 16∶0/20∶5, 18∶1/20∶5, 16∶0/22∶6 and 18∶1/22∶6 contributed 67.2% and 61.8% of PC and PE respectively but only 23.0% of PI. In PI the most important species was 18∶0/20∶4 at 36.7% but this species only constituted 0.7% in each of PC and PE. Small amounts of dipolyunsaturated species were also found in PC and PE. Molecular species are abbreviated as follows: e.g., 16∶0/20∶5 PC is 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine.     

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.     

Ozonation of PC in ethanol: separation and identification of a novel ethoxyhydroperoxide

The product of the ozonolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in ethanol-containing solvent was analyzed by chemiluminescence detection-HPLC with on-line electrospray MS, and characterized on the basis of NMR spectroscopy and MS in high-resolution fast atom bombardment mode. The reaction yielded a large amount of a novel ethoxyhydroperoxide compound [1-palmitoyl-2-(9-ethoxy-9-hydroperoxynonanoyl)-sn-glycero-3-phosphocholine]. In addition to a structural analysis, we speculate on the reaction pathway and discuss the possibility of ethoxyhydroperoxide as a potentially reactive ozonized lipid in food and biological materials.     

Molecular species composition of glycerophospholipids from white matter of human brain

The molecular species composition of the major glycerophospholipids from white matter of human brain were determined by high-performance liquid chromatography of the 3,5-dinitrobenzoyl derivatives of the corresponding diradylglycerols. In phosphatidylcholine (PC) and phosphatidylserine (PS), molecular species containing only saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) comprised 85.7 and 82.4% of the respective totals, with 18∶0/18∶1 predominant in PS and 16∶0/18∶1 in PC. These molecular species were also abundant in phosphatidylethanolamine (PE), but in this phospholipid species containing polyunsaturated fatty acids (PUFA), largely 18∶0/22∶6n−3 and 18∶0/20∶4n−6, accounted for over half the total; 18∶1/18∶1 was also abundant in PE. In contrast, 1-O-alk-1′-enyl-2-acylsn-glycero-3-phosphoethanolamine (GPE) had much more SFA- and MUFA-containing species, predominantly 16∶0a/18∶1, 18∶0a/18∶1 and 18∶1a/18∶1, with low amounts of species containing 20∶4n−6 and 22∶6n−3. In alkenylacyl GPE, 22∶4n−6 was the major PUFA and 16∶0a/22∶4n−6 and 18∶1a/22∶4n−6 the main PUFA-containing species. There was six times more 22∶6n−3, twice as much 20∶4n−6 and half the amount of 22∶4n−6 in PE as compared to alkenylacyl GPE. Molecular species are abbreviated as follows:e.g., 16∶0/18∶1 PE is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; the corresponding alkenylacyl species, 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine is 16∶0a/18∶1.     

Production of the criegee ozonide during the ozonation of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes

It is likely that Criegee ozonides are formed in small amounts in the lungs of animals breathing ozone-containing air. This makes these compounds potential candidates to act as secondary toxins which relay the toxic effects of ozone deeper into lung tissue than ozone itself could penetrate. Therefore, we have determined the yields of Criegee ozonides from unsaturated lipids in liposomal systems as a model of the types of yields of Criegee ozonides that might be expected both in the lung lining fluid layer and in biological membranes. Ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes produced bothcis- andtrans-Criegee ozonides. These ozonides have been isolated by solid phase extraction and high-performance liquid chromatography of the ozonized lipid, and the products have been identified by two-dimensional¹H nuclear magnetic resonance. The combined yield of thecis- andtrans-Criegee ozonides is 10.7±2.8% (avg. ±SD, n=7) with small unilamellar liposomes and 10.6±2.7% (n=3) with large multilamellar liposomes. We had previously reported (Chem. Res. Toxicol. 5 505–511, 1992) that ozonation of methyl oleate in sodium dodecylsulfate micelles also produces an 11% yield of the Criegee ozonides. Thus, ozonation in a variety of models gives about 11% of the Criegee ozonide, suggesting that these products also would be formed in small but significant amounts in the lungs of animals breathing polluted air. Further research on the pharmacokinetics and possible toxicity of the Criegee ozonides of fatty acids is suggested.     

Quantitative Analysis of Phospholipids Using Nanostructured Laser Desorption Ionization Targets

Since its introduction as an ionization technique in mass spectrometry, matrix-assisted laser desorption ionization (MALDI) has been applied to a wide range of applications. Quantitative small molecule analysis by MALDI, however, is limited due to the presence of intense signals from the matrix coupled with non-homogeneous surfaces. The surface used in nano-structured laser desorption ionization (NALDI) eliminates the need for a matrix and the resulting interferences, and allows for quantitative analysis of small molecules. This study was designed to analyze and quantitate phospholipid components of liposomes. Here we have developed an assay to quantitate the DPPC and DC_8,9PC in liposomes by NALDI following various treatments. To test our method we chose to analyze a liposome system composed of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DC_8,9PC (1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine), as DC_8,9PC is known to undergo cross-linking upon treatment with UV (254 nm) and this reaction converts the monomer into a polymer. First, calibration curves for pure lipids (DPPC and DC_8,9PC) were created using DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) as an internal standard. The calibration curve for both DPPC and DC_8,9PC showed an R² of 0.992, obtained using the intensity ratio of analyte and internal standard. Next, DPPC:DC_8,9PC liposomes were treated with UV radiation (254 nm). Following this treatment, lipids were extracted from the liposomes and analyzed. The analysis of the lipids before and after UV exposure confirmed a decrease in the signal of DC_8,9PC of about 90%. In contrast, there was no reduction in DPPC signal.     

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.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

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.     

Competitive deposition oftrans-12- andcis-9-octadecenoates into egg yolk lipids

The deposition oftrans-12-octadecenoate-12(13)-³H (12t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids.trans-12-Octadecenoate was preferentially incorporated into cholesteryl esters (CE), phosphatidylcholines (PC), and phosphatidylethanolamines (PE) but was discriminated against in triglycerides (TG). Isotopic ratios indicate that 5.9 and 5.6 times more 12t-18∶1-³H than 9c-18∶1-¹⁴C was esterified at the 1-acyl position of PE and PC, respectively. The combined 1- and 3-acyl positions of TG and the 2-acyl position of TG, PE and PC were each preferentially esterified with 9c-18∶1-¹⁴C.     

1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine content and molecular species composition in fish brain

Ethanolamine glycerophospholipids from the brains of both trout and cod comprised 36–38% of 1-O-alk-1′-enyl-2-acyl-glycerophosphoethanolamine (GPE) determined using two methods. In 1-O-alk-1′-enyl-2-acyl-GPE from trout brain, the main molecular species were 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1, which totalled 63.3%, while polyunsaturated fatty acid (PUFA) containing species totalled only 18.2%. 1-O-Alk-1′-enyl-2-acyl-GPE from cod brain was much more unsaturated with PUFA containing species totalling 52.6%, of which 18∶0a/20∶5n−3, 18∶1a/20∶5n−3 and 18∶1a/22∶6n−3 were predominant. In cod 18∶1a/18∶1, 18∶0a/18∶1 and 16∶0a/18∶1 were the only other species present at over 5% each, totalling 31.8%. In both cod and trout, small amounts of species containing 22∶4n−6 were found. The results of this and earlier studies indicate that there is considerable specificity of composition at the level of molecular species between different lipid classes and subclasses. Molecular species of 1-O-alk-1′-enyl-2-acyl-GPE are abbreviated as follows:e.g., 16∶0a/18∶1 GPE is 1-O-hexadec-1′-enyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. The corresponding diacyl species, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, is abbreviated as 16∶0/18∶1.     

Dietary fats containing concentrates ofcis ortrans octadecenoates and the patterns of polyunsaturated fatty acids of liver phosphatidylcholine and phosphatidylethanolamine

The effects of the mixedcis- 18∶1 isomers and mixedtrans-18∶1 isomers present in partially hydrogenated soybean oil (PHSO) upon the patterns of polyunsaturated fatty acids (PUFA) in liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in rats fed concentrates ofcis- 18∶1 ortrans- 18∶1 isomers isolated as triacylglycerides from PHSO. Thecis- 18∶1 andtrans- 18∶1 concentrates were fed at levels equal to those present in PHSO fed at 17.9% of the diet. All diets contained the required amounts of both linoleic and linolenic acids. Thetrans- 18∶1 concentrate was found to suppress the levels of 20∶4ω6 and 20∶3ω9, and to increase the levels of 18∶2ω6 and 20∶5ω3 in PC and PE. Thecis- 18∶1 concentrate suppressed 20∶4ω6 in PC, 20∶5ω3 in PC and PE, and 18∶2ω6 was more effective than thetrans concentrate in suppressing 22∶6ω3. Thetrans- 18∶1 concentrate was more effective in suppressing 20∶4ω6. Thetrans-18∶ isomers appear to modify PUFA metabolism by inhibition of PUFA synthesis, whereas thecis- 18∶1isomers appear to compete with 2-position fatty acyl transfer and to inhibit ω3 PUFA acylation.     

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl- and 1,2-diacyl glycerophospholipids in Japanese oysterCrassostrea gigas (Thunberg)

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl-, and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (EPL) andsn-glycero-3-phosphocholine (CPL) of Japanese oysterCrassostrea gigas were analyzed by selectedion monitoring gas chromatography/mass spectrometry using electron impact ionization. The characteristic fragment ions, [RCH=CH+56]⁺ due to the alkenyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkenylacylglycerols, [R+130]⁺ due to the alkyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkylacylglycerols, [RCO+74]⁺ due to the acyl residues in thesn-1 and/orsn-2 positions of diacylglycerols, and [M−57]⁺ being indicative of the corresponding molecular weight, were used for structural assignments. For alkenylacyl EPL and CPL, 19 and 16 molecular species were determined, respectively. Two molecular species, 18∶0alkenyl-22∶6n−3 and 18∶0-alkenyl-22∶2-non-methylene interrupted diene (NMID), amounted to 53.2% and 47.9%, respectively. The alkylacyl EPL and CPL consisted of 16 and 20 molecular species, respectively, and the prominent components were 18∶0alkyl-22∶2NMID, 20∶1alkyl-20∶1n−11 (27.4%) and 20∶1alkyl-20∶2NMID (16.3%) in the former, and 16∶0alkyl-20∶5n−3 (23.0%) and 16∶0alkyl-22∶6n−3 (21.6%) in the latter. For the diacyl EPL and CPL, 14 and 51 molecular species were determined, respectively. The major molecular species were 18∶0–20∶5n−3 (37.4%), 16∶0–20∶5n−3 (14.2%) and 18∶1n−7–22∶2NMID (13.2%) in the former, and 16∶0–20∶5n−3 (33.4%) and 16∶0–22∶6n−3 (22.3%) in the latter. It was found that there were significant differences in the molecular species between the alkylacyl and diacyl EPL and the alkylacyl and diacyl CPL; the number of molecular species was larger in CPL than in EPL, while the number of total carbons and double bonds of the major molecular species were larger in the EPL than in the CPL. Alkenylacyl EPL were similar to alkenylacyl CPL in molecular species composition.     

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride- or forskolin-stimulated activity in a murine fibroblast cell line,i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition ofcis-9-16∶1/cis-9-16∶1,cis-9-18∶1/cis-9-18∶1 orcis-9-18∶1/cis-9,12-18∶2sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, whilecis-11-18∶1/cis-11-18∶1 GPC was not effective and only a partial recovery was observed with 16∶0/16∶0, 16∶0/cis-9-18∶1 andtrans-9-18∶1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of thecis double bonds, each located in Δ9 position of the PC acyl chains. The limited effect ofcis-9-16∶1/cis-9-18∶1 GPC andcis-9-18∶1/cis-9-16∶1 GPC suggests that an equal length of the terminal hydrocarbon chains extending bevond the Δ9 double bonds is also important. Moreover, complete restoration of adenylate cyclase activity in cells exposed to 16∶0/cis-9,12-18∶2 GPC suggests that twocis-9,12 double bonds located on the same chain are as effective as twocis-9 double bonds each located on two different chains of PC. As the four double bonds of 16∶0/cis-5,8,11,14-20∶4 GPC had no effect, a mere increase in the number of double bonds seems insufficient to build an optimal lipid microenvironment for the enzyme.     

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.