Determination of phosphatidylcholine monohydroperoxides using quadrupole time-of-flight mass spectrometry

An improved technique for the analysis of phosphatidylcholine (PC) monohydroperoxides was developed using quadrupole time-of-flight (Q-TOF) mass spectrometry with electrospray ionization. Separation was obtained using an HPLC C8 column with a gradient of methanol and 10 mM aqueous ammonium acetate. Monohydroperoxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH(+) and [M+Na](+) ions in the heart of the intact rat. Using standard synthetic PCOOH (C16:0/C18:2-OOH), the lipid extract component was identified as (C16:0/C18:2-OOH) PC based on the product ions of ESI-MS-MS and, the PCOOH concentration was quantitated using HPLC with chemiluminescence detection. Two epoxyhydroxy derivatives of the three PCs mentioned above were also detected. This is the first report to show the presence of monohydroperoxides and epoxyhydroxy-derivatives of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

PI3K–Akt signaling controls PFKFB3 expression during human T-lymphocyte activation

It is commonly accepted that brain phospholipids are highly enriched with long-chain polyunsaturated fatty acids (PUFAs). However, the evidence for this remains unclear. We used HPLC–MS to analyze the content and composition of phospholipids in rat brain and compared it to the heart, kidney, and liver. Phospholipids typically contain one PUFA, such as 18:2, 20:4, or 22:6, and one saturated fatty acid, such as 16:0 or 18:0. However, we found that brain phospholipids containing monounsaturated fatty acids in the place of PUFAs are highly elevated compared to phospholipids in the heart, kidney, and liver. The relative content of phospholipid containing PUFAs is ~ 60% in the brain, whereas it is over 90% in other tissues. The most abundant species of phosphatidylcholine (PC) is PC(16:0/18:1) in the brain, whereas PC(18:0/20:4) and PC(16:0/20:4) are predominated in other tissues. Moreover, several major species of plasmanyl and plasmenyl phosphatidylethanolamine are found to contain monounsaturated fatty acid in the brain only. Overall, our data clearly show that brain phospholipids are the least enriched with PUFAs of the four major organs, challenging the common belief that the brain is highly enriched with PUFAs.     

Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing

In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [(14)C]acetate, [(14)C]glycerol, and [(14)C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [(14)C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [(14)C]18:1 and [(14)C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [(14)C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation.     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.     

Differential methylation patterns in molecular species of phosphatidylethanolamine derivatives in rat liver membranes

The appearance of individual molecular species of phospholipids in the complete sequence of the transmethylation of phosphatidylethanolamine (PE) was examined in rat liver microsomes incubated with S-adenosyl-L-[methyl-14C]methionine. Reverse-phase HPLC analysis of phosphatidylcholine (PC), phosphatidyl-N,N-dimethylethanolamine (dimethyl-PE), or phosphatidyl-N-monomethylethanolamine (monomethyl-PE) showed that radioactivity was present in the same six principal molecules; a first group is constituted by 16:0/22:6, 16:0/20:4 and 16:0/18:2 and a second one by the homologous molecules with 18:0 instead of 16:0 at the sn-1 position of glycerol. In PC, 16:0/22:6 (23% of total radioactivity) was preponderant, and 18:0/20:4 was the lowest. The ratios cpm in PC/nmol in PE were in the order: 16:0/22:6 greater than 16:0/18:2 greater than 16:0/20:4 followed by the corresponding 18:0 molecules. On the other hand, in intermediate phospholipids, incorporation of methyl groups was most marked in 18:0/20:4 (24-27% of total). 16:0/22:6 and 16:0/18:2 were low in comparison to their relative values in PC. The ratio (18:0/20:4)/(16:0/22:6) was 4.5-5.6-times higher in monomethyl-PE and dimethyl-PE than in PC. These differences were found consistently, regardless of incubation time of microsomes (2.5-60 min) and of S-adenosyl-L-methionine (AdoMet) concentration (3 or 100 microM). In liver membranes, it would therefore seem that there is a different selectivity in methyl group transfer, depending upon whether the first two steps or the third step of the reaction are considered. Side reactions, such as deacylation/reacylation, are unlikely to account for this difference, which could rather be related to the enzyme itself.     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Analyses for phosphatidylcholine hydroperoxides by LC/MS

A new liquid chromatography mass spectrometry (LC/MS) method has been developed for the qualitative and quantitative analyses of phosphatidylcholine hydroperoxides (PC-OOH) in human plasma using a synthetic hydroperoxide (1-stearoyl-2-erucoyl-PC monohydroperoxide, PC 18:0/22:1-OOH) as an internal standard. 1-Stearoyl-2-linoleoyl-PC monohydroperoxide (PC 18:0/18:2-OOH) was identified in plasma by LC/MS by comparison with an authentic standard. The calibration curves obtained for 1-palmitoyl-2-linoleoyl-PC monohydroperoxide, PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were linear throughout the calibration range (0.1–1.0 pmol). The limit of detection (LOD) (S/N = 3:1) was 0.01 pmol, and the limit of quantification (LOQ) (S/N = 6:1) was 0.1 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 89 and 32 nM, respectively, in a healthy volunteer.     

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.     

Fatty acid composition of lipid A in Pseudomonas fluorescens

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.     

An improved HPLC assay for phosphatidylcholine hydroperoxides (PCOOH) in human plasma with synthetic PCOOH as internal standard

Quantitative and qualitative analyses of 1-palmitoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 16:0/18:2-OOH] and 1-stearoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 18:0/18:2-OOH] in human plasma were improved by chemiluminescence HPLC using synthetic 1-stearoyl-2-erucoyl-phosphatidylcholine monohydroperoxide (PC 18:0/22:1-OOH) as internal standard. The calibration curves of synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, obtained by their direct injections with the IS into the HPLC system, were linear throughout the calibration range (10-1000 pmol). Within-day and between-day coefficients of variation were below 8%, and the recoveries were between 84% and 101%. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 102+/-59 nM (mean+/-SD) and 36+/-20 nM, respectively, in the 33 healthy volunteers. The present method might help understanding incompletely understood pathway of plasma phosphatidylcholine hydroperoxides.     

Trans unsaturated fatty acids are less oxidizable than cis unsaturated fatty acids and protect endogenous lipids from oxidation in lipoproteins and lipid bilayers

Epidemiological data suggest that dietary trans unsaturated fatty acids increase the risk of heart disease; however, the underlying mechanisms are unclear. In this study, we investigated one possible mechanism, namely, their effect on LDL oxidation. Supplementation of LDL with 10% 16:1 trans-cholesteryl ester (CE) inhibited the oxidation compared to that with 16:1 cis-CE. Total replacement of core lipids with 18:2 trans,trans-CE decreased the rate of LDL oxidation by 19% compared to replacement with 18:2 cis,cis-CE. When the surface phosphoglycerides were replaced with either 16:0-18:2 cis,cis-phosphatidylcholine (PC) or 16:0-18:2 trans,trans-PC, the latter was found to inhibit the rate and increase the lag time of oxidation to a greater extent than the former. To confirm these findings, we studied the oxidation of PC liposomes by assessing the formation of conjugated dienes or the degradation of a fluorescently labeled PC. By both methods, the 16:0-18:2 trans,trans-PC exhibited greater resistance to oxidation than the 16:0-18:2 cis,cis-PC. Eliminating the fluidity differences did not completely eliminate the differences in oxidation rates, suggesting that the trans double bond is inherently resistant to oxidation. The composition of the conjugated hydroperoxy products formed after oxidation differed markedly for the two 18:2 isomers. Supplementation of 16:0-18:2 cis,cis-PC liposomes with 20 mol % di16:1 trans-PC retarded oxidation rates to a greater extent than supplementation with di16:1 cis-PC. These studies show that dietary trans unsaturated fatty acids decrease the rate of lipid peroxidation, an effect that may mitigate the atherogenic effect of these fatty acids.     

Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster)

The total lipids of muscle and cephalothorax of Mediterranean lobster Palinurus vulgaris were found to be 1.0% and 2.4% of the wet tissue of which the phospholipids represented 66.5% and 47.5%, respectively. The main PhL saturated, monounsaturated and polyunsaturated fatty acids in muscle and cephalothorax were C16:0, C18:0, C18:1ω-9, C18:1ω-7, C20:4ω-6, C20:5ω-3 and C22:6ω-3. 2-OH C14:0 and cyclo-17:0 fatty acids were also identified though in low percentages. The main individual PhL in muscle were found to be phosphatidylcholine (53.5%), 72.0% of which corresponded to the structure of 1,2-diacyl-glycerocholine while the rest 28.0% to 1-O-alkyl-2-acyl-glycerocholine or 1-O-(1-alkenyl)-2-acyl-glycerocholine and phosphatidylethanolamine (19.3%), 75.0% of which corresponded to the structure of 1,2-diacyl-glyceroethanolamine and 25% to 1-O-alkyl-2-acyl-glyceroethanolamine or 1-O-(1-alkenyl)-2-acyl-glyceroethanolamine. Cephalothorax main PhL were found to be PC and PE (66.4% and 18.8%, respectively). In muscle and cephalothorax PC ω-3 fatty acids amounted 7.78% and 8.60%, while in PE amounted 30.77% and 23.65% respectively. Furthermore, in both tissues PhL, cardiolipine phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatidylcholine, were also found.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Impact of polyunsaturated fatty acid degradation on survival and acidification activity of freeze-dried <Emphasis Type="Italic">Weissella paramesenteroides</Emphasis> LC11 during storage

The impact of polyunsaturated fatty acid (PUFA) degradation on the survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 was investigated over 90-days storage at 4 degrees C or 20 degrees C in vacuum-sealed aluminium foil or glass tubes with two water activities (a(w)=0.11 or 0.23). Colony counts, acidification activity (% lactic acid/g), linoleic/palmitic (18:2/16:0) or linolenic/palmitic (18:3/16:0) ratio by gas chromatography and 18:2 or 18:3 oxylipins by reversed phase-high performance liquid chromatography were determined. The viable cells, acidification activity and 18:2/16:0 or 18:3/16:0 ratio decreased as the storage time increased. The survival, acidification activity and 18:2/16:0 or 18:3/16:0 ratio were greatest for the freeze-dried strain held in vacuum-sealed aluminium foil at 4 degrees C. The 18:2/16:0 or 18:3/16:0 ratio decrease was correlated with the accumulation of 18:2 or 18:3 oxylipins during storage in glass tubes. Hydroperoxy PUFAs, hydroxy PUFAs, divinyl ether PUFAs and oxo PUFAs were the main oxylipins identified. A large decrease in the 18:2/16:0 or 18:3/16:0 ratio and a rapid accumulation of oxylipins during storage might be enough to cause high cell death and loss of metabolic activity. These results provide further experimental support for the hypothesis that lipid oxidation and survival or activity of freeze-dried bacteria might be related.     

Analysis of effector cell-derived lyso platelet activating factor by electron capture negative ion mass spectrometry.

Quantification of 1-O-alkyl-2-lyso-sn-3-glycero-phosphocholine (lysoPAF) and determination of the different molecular species released by cells has been hampered by the molecules's lack of intrinsic bioactivity, unavailability of a suitable internal standard, and reliance on derivatives requiring electron impact techniques. We have synthesized trideuterated internal standards (labeled on the terminal carbon of the alkyl chain) for both C16:0 and C18:0 lysoPAF. Using these standards, we isolated and quantified lysoPAF released from A23187-stimulated human neutrophils and rat alveolar macrophages. Extracted lysoPAF was purified by solid-phase extraction and thin-layer chromatography. The polar phosphorylcholine group was removed with 29 M HF or phospholipase C. The two free hydroxyl groups were derivatized with pentafluorobenzoyl chloride. The resultant bis-pentafluorobenzoyl derivative, analyzed by gas chromatography/electron capture negative ion mass spectrometry, underwent substantial fragmentation. Lowering of the ion source temperature resulted in a dramatic increase in signal-to-noise ratio, with the vast majority of the ion current carried in the molecular anion. Stimulated neutrophils released 16.3 and 10.2 ng/10(6) cells of C16:0 lysoPAF and C18:0 lysoPAF, respectively. Rat macrophages synthesized 15.9 ng/10(6) cells of C16:0 lysoPAF, but C18:0 lysoPAF was variably detected at low levels. We conclude that use of the bispentafluorobenzoyl ester derivative of lysoPAF allows facile quantification of this autacoid metabolite in biological matrices.     

Enterobacter soli sp. nov.: A Lignin-Degrading ��-Proteobacteria Isolated from Soil

A Gram-negative bacterium that formed cream-colored colonies designated strain LF7 was isolated from soil collected in the Tambopata National Reserve in Madre de Dios, Peru. 16S rRNA sequence comparisons indicate that LF7 is a novel Enterobacter sp. closely related to E. asburiae JCM 6051(T) [AB004744] and E. aerogenes JCM 1235(T) [AB004750] based on their sequence homologies (p-distance: 1.06 and 1.19%, respectively). DNA G + C content was 52.8 mol% which is within the range reported for E. asburiae (55-57 mol%). The major cellular fatty acids present in the LF7 strain were C(16:0) (27.3%), C(16:1) ω7c and/or C(16:1) ω6c (16.3%), C(18:1) ω7c (16.1%), C(17:0) cyclo (12.4%), C(14:0) 3-OH and/or C(16:1) iso-I (8.9%), C(14:0) (7.6%), C(12:0) (3.9%), C(17:0) (2.4%), C(13:0) 3-OH and/or C(15:1) iso-H (1.7%), C(13:0) (1.1%), and C(18:2) ω6,9c and/or C(18:0) ante (0.5%). The cellular fatty acid profile, G + C content, phenotypic and biochemical characteristics were consistent with its placement in the genus Enterobacter. The name Enterobacter soli is proposed for this bacterium.     

Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.     

Species-specific variations in the molecular heterogeneity of the platelet-activating factor

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.     

Fatty acid exchange of phospholipids in membranes of rat heart

Incorporation of ¹⁴C fatty acids in phospholipids of plasma membranes and sarcoplasmic reticulum of rat heart was studied. Mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were labelled. Our studies showed that the incorporation of unsaturated fatty acids (oleic and linoleic acid) was higher than for saturated fatty acids (palmitic and stearic acid). The range of uptake was between 0.2 and 2.2 nmol·mg⁻¹ protein·h⁻¹ and 0.5–7.4 nmol·μgatom⁻¹ P₁·h⁻¹, respectively. Uptake of activity in individual phospholipids (measured after separation on TLC) was calculated as percentage of total activity. Incorporation in phosphatidylcholine was higher than in phosphatidylethanolamine. Phosphatidylcholine showed an increasing sequence for the following fatty acids: C_18:0 < C_16:0 < C_18:0 < C_18:2. However, phosphatidylethanolamine showed a decreasing sequence for the incorporation of the same fatty acids. Labelling of PC was always greater than for PE, except for stearic acid which was better incorporated into phosphatidylethanolamine. Uptake of the same fatty acid into phospholipids of sarcoplasmic reticulum was always higher than uptake into plasma membranes. As incorporation of fatty acids bound to albumin was studied in isolated membranes of rat heart, the addition of ATP and CoASH was an absolute requirement.