Polyunsaturated Molecular Species Research Articles

LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

Effects of microwave vs. convection oven heating on the formation of oxidation products in canola (Brassica rapa subsp. oleifera) oil

Research on the effects of microwave vs. “conventional” heating of dietary oils on lipid oxidation has been very limited. In this study, canola oil (Brassica rapa subsp. oleifera ) was heated in either convection or microwave oven to compare the effects of heating methods on triacylglycerol (TAG) oxidation. Peroxide and p -anisidine values (PV and p -AV, respectively) were determined and liquid chromatography–mass spectrometric (LC–MS) analysis of non-oxidized and oxidized TAG molecular species was performed. Neither of the heat treatments caused any considerable changes in PV of the oil samples. However, increase in p -AV was observed. The change was higher in the oil heated in microwave oven, demonstrating a higher increase in the amount of secondary oxidation products. The changes were accompanied by a decrease in the polyunsaturated TAG molecular species ACN:DB (acyl carbon number:number of double bonds) 54:7 and 54:6, this change also being higher in the oil heated in microwave oven.

Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro

The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane’s composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the development of novel strategies to modulate oocyte membrane dynamics and structure.

Polyunsaturated lysophosphatidic acid as a potential asthma biomarker.

Lysophosphatidic acid (LPA), a lipid mediator in biological fluids and tissues, is generated mainly by autotaxin that hydrolyzes lysophosphatidylcholine to LPA and choline. Total LPA levels are increased in bronchoalveolar lavage fluid from asthmatic lung, and are strongly induced following subsegmental bronchoprovocation with allergen in subjects with allergic asthma. Polyunsaturated molecular species of LPA (C22:5 and C22:6) are selectively synthesized in the airways of asthma subjects following allergen challenge and in mouse models of allergic airway inflammation, having been identified and quantified by LC/MS/MS lipidomics. This review discusses current knowledge of LPA production in asthmatic lung and the potential utility of polyunsaturated LPA molecular species as novel biomarkers in bronchoalveolar lavage fluid and exhaled breath condensate of asthma subjects.

Polyunsaturated molecular species of galactolipids: Markers of zooxanthellae in a symbiotic association of the soft coral Capnella sp. (Anthozoa: Alcyonacea)

A method that uses marker fatty acids (FAs) is widely applied in investigations of trophic and symbiotic relationships. In a search for new lipid markers, we determined the total lipid FA composition, as well as the composition of molecular species of mono- and digalactosyl diacylglycerols (MGDGs and DGDGs), which are specific galactolipids of thylakoid membranes, in zooxanthellae (endosymbiotic dinoflagellates) of the tropical soft coral Capnella sp. Some FAs of zooxanthellae were suggested for use as marker polyunsaturated FAs (PUFAs). Thirteen molecular species of MGDGs and ten molecular species of DGDGs were detected using the method of high-resolution tandem mass spectrometry. All marker PUFAs of zooxanthellae were found in acyl groups of galactolipids. The major molecular species of DGDGs (18:4/18:4, 18:4/20:5, and 16:2/22:6) and the unique molecular species of MGDGs (16:4/18:5) were described. The identification of several polyunsaturated molecular species of galactolipids that contain marker FAs allowed us to propose that this lipid group be used as molecular lipid markers of zooxanthellae for the study of symbiont–host interactions in soft corals.

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.

A mitochondrial pathway for biosynthesis of lipid mediators.

The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury.

高度不飽和グリセロ脂質の分子種の分析と機能に関する研究

逆相分配クロマトグラフィーにおける高度不飽和グリセロ脂質分子種の保持容量 (保持時間) を支配する因子, および隣り合うグリセロ脂質分子種の分離度を支配する因子を明らかにし, それらを応用することによって水産グリセロ脂質の内, とくにリン脂質の分子種を同定し, その特徴を明らかにした。次いで, 水産グリセロリン脂質の主要分子種であるDHA結合型リン脂質分子種を合成し, その有用機能の一端として抗腫瘍性物質に対する機能促進性を明らかにするとともに, それを応用したリボソームも作成し, その抗腫瘍性からみた有用性についても評価した。

Caloric Restriction Results in Phospholipid Depletion, Membrane Remodeling, and Triacylglycerol Accumulation in Murine Myocardium

Herein, we utilize the power of shotgun lipidomics to demonstrate that modest caloric restriction results in phospholipid depletion, membrane remodeling, and triacylglycerol (TAG) accumulation in murine myocardium. After brief periods of fasting (4 and 12 h), substantial decreases occurred in the choline and ethanolamine glycerophospholipid pools in murine myocardium (collectively, a decrease of 39 nmol of phospholipid per milligram of protein at 12 h representing approximately 25% of total phospholipid mass and approximately 20 cal of Gibbs free energy per gram wet weight of tissue). Remarkably, the selective loss of long-chain polyunsaturated molecular species was present in the major phospholipid classes thereby altering the physical properties of myocardial membranes. No decrease in TAG mass was present in myocardium during fasting, but rather myocardial TAG increased during 12 h of refeeding nearly 3-fold returning to baseline levels only after 24 h of refeeding. No alterations in other examined lipid classes were present during fasting. In contrast to these lipid alterations in myocardium, no decreases in phospholipid mass were present in skeletal muscle myocytes and a dramatic decrease in skeletal muscle (or skeletal muscle associated) TAG mass was prominent after 12 h of fasting. These results identify phospholipids as a rapidly mobilizable energy source during modest caloric deprivation in murine myocardium, while triacylglycerols are a major source of energy reserve in skeletal muscle. Collectively, these results demonstrate dramatic alterations in the membrane composition of mildly fasted mammalian myocardium that identify the unanticipated plasticity of myocardial phospholipids to adapt to modest chemical and physical perturbations.

Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry.

Phospholipids (PL) in cerebral cortex from patients with infantile (INCL or CLN1) and juvenile (JNCL or CLN3) forms of neuronal ceroid-lipofuscinosis (NCL) and controls were analysed by normal phase HPLC and on-line electrospray ionization ion-trap mass spectrometric detection (LC-ESI-MS). The method provided quantitative data on numerous molecular species of different PL classes, which are not achieved by using the conventional chromatographic methods. Compared with the controls, the INCL brains contained proportionally more phosphatidylcholine (PC), and less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Different molecular species of PC, PE, PS, phosphatidylinositol and sphingomyelin were quantified using multiple internal PL standards that differed in fatty acyl chain length and thus allowed correction for chain length dependency of instrument response. In INCL cortex, which had lost 65% of the normal PL content, the proportions of polyunsaturated molecular species, especially the PS and PE that contained docosahexaenoic acid (22:6n-3), were dramatically decreased. The membranes may have adapted to this alteration by increasing the proportions of PL molecules substituted with monounsaturated and short-chain fatty acids. Lysobisphosphatidic acid was highly elevated in the INCL brain and consisted mostly of polyunsaturated species. It is possible that changes in the composition of PL membranes accelerate progression of INCL by altering signalling and membrane trafficking in neurons.

Metabolic alterations by clofibric acid in the formation of molecular species of phosphatidylcholine in rat liver

The mechanism by which p-chlorophenoxyisobutyric acid (clofibric acid) induces striking changes in the proportion of the molecular species of phosphatidylcholine (PC) in rat liver was studied. Treatment of rats with clofibric acid strikingly increased the content of 1-palmitoyl-2-oleoyl (16:0–18:1) PC, but decreased the contents of 1-palmitoyl-2-docosahexaenoyl (16:0–22:6), 1-stearoyl-2-arachidonoyl (18:0–20:4), and 1-stearoyl-2-linoleoyl (18:0–18:2) PC; the drug did not change the content of 1-palmitoyl-2-arachidonoyl (16:0–20:4) PC. The mechanism underlying these changes has been investigated with regard to the in vivo formation of the molecular species of PC by: (i) de novo synthesis, (ii) reacylation, and (iii) methylation of phosphatidylethanolamine (PE). We found that (i) the incorporation of [3H]glycerol, which was injected intravenously, into 16:0–18:1 diacylglycerol (DG) and 16:0–18:1 PC was increased markedly by clofibric acid feeding without changing the substrate specificity of CDP-choline:DG cholinephosphotransferase, (ii) the in vivo formation of 16:0–18:1 and 16:0–20:4 PC from 1–16:0-[3H]glycerophosphocholine (GPC), which was injected intraportally, was increased markedly by clofibric acid feeding, and (iii) the incorporation of [14C]ethanolamine, which was injected intravenously into 16:0–22:6, 18:0–22:6, and 18:0–20:4 PC, was decreased by clofibric acid feeding; the extent of the decrease in 16:0–20:4 PC was less than that of 18:0–20:4 PC. It was concluded, therefore, that (i) clofibric acid selectively increased the content and proportion of 16:0–18:1 PC by enhancing both the CDP-choline pathway and the remodeling of the pre-existing PC molecule, and (ii) the drug kept the content of 16:0–20:4 PC unchanged by stimulating the remodeling of the pre-existing PC molecule, whereas the formation of other more long chain, polyunsaturated molecular species, such as 16:0–22:6, 18:0–22:6, and 18:0–20:4, was decreased owing to the suppression of PE methylation.

The effect of low alpha-linolenic acid diet on glycerophospholipid molecular species in guinea pig brain.

The changes in guinea pig brain (cerebrum) glycerophospholipid molecular species resulting from a low-alpha linolenic acid (ALA) diet are described. Two groups of six guinea pigs were raised from birth to 16 wk of age on either an n-3 deficient diet containing 0.01 g ALA/100 g diet or n-3 sufficient diet containing 0.71 g ALA/100 g diet. Molecular species of diradyl glycerophosphoethanolamine (GroPEtn), glycerophosphocholine, glycerophosphoserine, and glycerophosphoinositol were analyzed by high-performance liquid chromatography with on-line electrospray ionization mass spectrometry (HPLC/ESI/MS). Alkenylacyl GroPEtn species were determined by comparing spectra before and after mild acid treatment while diacyl- and alkylacyl species were distinguished by HPLC/ESI/MS. The proportions of phospholipid classes and of the diradyl GroPEtn subclasses were not altered by diet changes. The main polyunsaturated molecular species of diradyl GroPEtn subclasses and of phosphatidyl choline and phosphatidylserine (PtdSer) contained 16:0, 18:0, or 18:1 in combination with docosahexaenoic acid (DHA, 22:6n-3), docosapentaenoic (DPA, 22:5n-6), or arachidonic acid (ARA, 20:4n-6). A significant proportion of DPA containing species were present in both diet groups, but in n-3 fatty acid deficiency, the proportion of DPA increased and DHA was primarily replaced by DPA. The combined value of main DHA and DPA containing species in the n3 deficient group ranged from 91-111% when compared with the n-3 sufficient group, indicating a nearly quantitative replacement. The n-3 fatty acid deficiency did not lower the content of ARA containing molecular species of PtdSer of the guinea pig brain as reported previously for the rat brain. The molecular species of phosphatidylinositol were not altered by n-3 fatty acid deficiency. The present data show that the main consequence of a low ALA diet is the preferential replacement of DHA-containing molecular species by DPA-containing molecular species in alkenylacyl- and diacyl GroPEtn and PtdSer of guinea pig brain.

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver.

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids.

Freshly isolated rat hepatocytes were incubated for 20 min with [U-14C]glycerol in the presence or absence of unlabeled linoleic (18:2n-6), arachidonic (20:4n-6), or docosahexaenoic (22:6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (approximately 95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-14C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U- 14C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U- 14C]glycerol and by determining incorporation of 18O from H 2 18O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U- 14C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca 2+-dependent phospholipase A 2 activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.

High-performance liquid chromatographic method for determination of the metabolism of polyunsaturated molecular species of phosphatidylserine labeled in the polar group.

A reversed-phase HPLC method to monitor the incorporation of radiolabeled precursors into the polar group of individual polyunsaturated molecular species of phosphatidylserine (PS) is presented. PS labeled in the polar group was decarboxylated and subsequently converted to trinitrophenyl-phosphatidylethanolamine (Tnp-PE), which was separated into its molecular species by reversed-phase HPLC within 90 min, using a gradient of acetonitrile-methanol and ammonium acetate. A major feature of the method is the complete resolution of the stearoyl species, 18:0/20:4 and 18:0/22:6, at ambient temperature. By determining the amount of radioactivity incorporated into each fraction, the metabolism of individual molecular species of PS, and also of PE, labeled in the polar group can be investigated.

Ischemia-induced changes in cerebral mitochondrial free fatty acids, phospholipids, and respiration in the rat.

Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.

Mechanisms of synthesis of polyunsaturated phosphatidylcholine molecular species by guinea pig liver.

Mechanisms of synthesis of polyunsaturated phosphatidylcholine molecular species by guinea pig liver.

Acylation of alkyllysophospholipids by Fischer sarcoma microsomes

Acylation of alkyllysophospholipids in most cells occurs by: (a) CoA-independent transacylation, (b) CoA-dependent transacylation, and (c) acyl-CoA-dependent acylation. Using a recently developed high-performance liquid chromatography method, we have investigated the factors that influence the molecular species composition of the acylated products formed via these pathways with 1-hexadecyl-2-lyso- sn-glycero-3-phosphocholine (alkyl-lyso-GPC) or 1-hexadecyl-2-lyso- sn-glycero-3-phosphoethanolamine (alkyllyso-GPE) as substrates for the enzymes in Fischer R-3259 sarcoma microsomes. We found that short incubation times and low substrate concentrations favored the formation of polyunsaturated molecular species, i.e., 16:0–22:6,16:0–22:5 ( n − 3), and 16:0–20: 4. Also, in agreement with results from other systems, CoA-independent transacylation produced a high percentage of polyunsaturated molecular species; acyl-CoAdependent acylations generated the least polyunsaturated molecular species and CoA-dependent transacylation gave intermediate values. Furthermore, no substrate selectivity occurred with respect to alkyl chain lengths of alkyllyso-GPE; similar molecular species composition was obtained with either hexadecyllyso-GPE or octadecyllyso-GPE as substrates. Responses to N-ethylmaleimide inhibition and heat inactivation as well as pH optima suggest the same enzyme catalyzes the CoA-independent transacylation of both alkyllyso-GPC and alkyllyso-GPE.