Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.     

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.     

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.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design

The thermoalkalophilic lipase from Bacillus thermocatenulatusBTL2 exhibits a low phospholipase activity (lecithin/tributyrinratio 0.03). A single round of random mutagenesis of the BTL2gene followed by screening of 6000 transformants on egg-yolkplates identified three variants with 10–12-fold increasedphospholipase activities, corresponding to lecithin/tributyrinratios of 0.16–0.36. All variants were specific for thesn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.Mutations occurred predominantly in the N-terminal part of BTL2with regions surrounding the predicted helix     

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.     

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.     

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.     

Diacylglycerol acyltransferases from Vernonia and Stokesia prefer substrates with vernolic acid

Genetic engineering of common oil crops for industrially valuable epoxy FA production by expressing epoxygenase genes alone had limited success. Identifying other key genes responsible for the selective incorporation of epoxy FA into seed oil in natural high accumulators appears to be an important next step. We investigated the substrate preferences of acyl CoA: diacylglycerol acyltransferases (DGAT) of two natural high accumulators of vernolic acid, Vernonia galamensis and Stokesia laevis, as compared with a common oilseed crop soybean. Developing seed microsomes were fed with either [¹⁴C]oleoyl CoA or [¹⁴C]vernoloyl CoA in combinations with no exogenous DAG or with 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-vernoloyl-sn-glycerol, 1,2-divernoloyl-sn-glycerol, 1,2-dioleoyl-rac-glycerol, or 1,2-divernoloyl-rac-glycerol to determine their relative incorporation into TAG. The results showed that in using sn-1,2-DAG, the highest DGAT activity was from the substrate combination of vernoloyl CoA with 1,2-divernoloyl-sn-glycerol, and the lowest was from vernoloyl CoA or oleoyl CoA with 1,2-dioleoyl-sn-glycerol in both V. galamensis and S. laevis. Soybean DGAT was more active with oleoyl CoA than vernoloyl CoA, and more active with 1,2-dioleoyl-sn-glycerol when oleoyl CoA was fed. DGAT assays without exogenous DAG, or with exogenous sn-1,2-DAG fed individually or simultaneously showed consistent results. In combinations with either oleoyl CoA or vernoloyl CoA, DGAT had much higher activity with rac-1,2-DAG than with their corresponding sn-1,2-DAG, and the substrate selectivity was diminished when rac-1,2-DAG were used instead of sn-1,2-DAG. These studies suggest that DGAT action might be an important step for selective incorporation of vernolic acid into TAG in V. galamensis and S. laevis.     

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.     

The degradation of platelet-activating factor and related lipids: Susceptibility to phospholipases C and D

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) is an ether-linked lipid that exhibits selective cytotoxicity toward several types of tumor cells and is relatively inactive toward normal cells under the same conditions of treatment. The mechanism of this selective cytotoxicity is unknown. We conducted studies to determine whether this compound is metabolized by phospholipases C and D and, if so, whether sensitive and resistant cells differ in their ability to degrade ET-18-OCH₃ by these enzymes. We have examined the metabolism of the L-isomer of ET-18-OCH₃, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH₃), by lysophospholipase D of rat liver microsomes and by a phospholipase D from the marine bacteriumVibrio damsela. The metabolism of L-ET-18-OCH₃ was also examined in cell culture using Madin-Darby canine kidney cells, human promyelocytic leukemia cells and human myelocytic leukemia cells. In these studies, L-ET-18-OCH₃ and related 1-O-alkyl-linked phosphocholine analogs radiolabeled with³H in the 1-O-alkyl chain were used. L-ET-18-OCH₃ was not hydrolyzed by lysophospholipase D from rat liver microsomes under conditions where cleavage of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine was observed. However, phospholipase D from the marine bacteriumV. damsela readily hydrolyzed L-ET-18-OCH₃ to 1-O-[³H]octadecyl-2-O-methyl-sn-glycero-3-phosphate, demonstrating that L-ET-18-OCH₃ can be degraded by a phospholipase D. Platelet-activating factor (PAF) and lyso-PAF were also substrates for the bacterial phospholipase D. When intact cells were incubated with radiolabeled L-ET-18-OCH₃ a product was formed that was identified as 1-O-[³H]octadecyl-2-O-methyl-sn-glycerol. There are two mechanisms that could account for the appearance of this product. The first involves cleavage of the compound by a phospholipase C, resulting in direct release of the diglyceride. The second possible mechanism involves cleavage by a phospholipase D to form the phosphatidic acid analog with subsequent hydrolysis to the diglyceride by a phosphohydrolase. Preliminary data support the phospholipase C-type mechanism. Regardless of which mechanism operates in intact cells, the metabolic degradation of L-ET-18-OCH₃ does not appear to be a significant factor in the selective cytotoxicity of this antitumor agent.     

Asymmetric <Emphasis Type="Italic">in vitro</Emphasis> synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30°C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30–40% 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) and 60–70% 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3′ position of the glycerol molecule.     

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.     

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.     

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.     

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.     

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0–60°C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-dioleoyl-sn-glycero-3-phosphol-1′-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50–60°C to 70% R,R and 30% R,S at O°C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65–69% R,R in the temperature range of 60–10°C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40°C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.     

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.     

Synthesis of C-18 mixed acid diacyl-sn-glycerol enantiomers

Procedures have been developed for the synthesis of both enantiomeric forms of mixed fatty acid, saturated and polyunsaturated 1,2-diacyl-sn-glycerols and 2,3-diacyl-sn-glycerols from D-mannitol as starting material. The following diacyl-sn-glycerols have been synthesized: 1-Stearoyl-2-linoleoyl-sn-glycerol, 1-stearoyl-2-linolenoyl-sn-glycerol, 2-linoleoyl-3-stearoyl-sn-glycerol and 2-linolenoyl-3-oleoyl-sn-glycerol. Their specific rotations, refractive indices, densities, solubilities, carbon and hydrogen analysis and iodine values have been reported. Presented in part at the ISF-AOCS World Congress, Chicago, September 1970.     

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.