Ethanolamine Glycerophospholipids Research Articles

Quantitative analysis of phospholipids containing arachidonate and docosahexaenoate chains in microdissected regions of mouse brain

Phospholipids containing polyunsaturated fatty acyl chains are prevalent among brain lipids, and regional differences in acyl chain distribution appear to have both functional and pathological significance. A method is described in which the combined application of GC and multiple reaction monitoring (MRM) MS yielded precise relative quantitation and approximate absolute quantitation of lipid species containing a particular fatty acyl chain in milligram-sized tissue samples. The method uses targeted MRM to identify specific molecular species of glycerophosphocholine lipids, glycerophospho-ethanolamine lipids, glycerophosphoinositol lipids, glycerophosphoserine lipids, glycero-phosphoglycerol lipids, and phosphatidic acids that contain esterified arachidonate (AA) and docosahexaenoate (DHA) separated during normal phase LC/MS/MS analysis. Quantitative analysis of the AA and DHA in the LC fractions is carried out using negative ion chemical ionization GC/MS and stable isotope dilution strategies. The method has been applied to assess the glycerophospholipid molecular species containing AA and DHA in microdissected samples of murine cerebral cortex and hippocampus. Results demonstrate the potential of this approach to identify regional differences in phospholipid concentration and reveal differences in specific phospholipid species between cortex and hippocampus. These differences may be related to the differential susceptibility of different brain regions to neurodegenerative disorders.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

Genetic ablation of calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA(2)gamma(-/-) mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA(2)gamma in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA(2)gamma loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

Analysis of Phospholipids in Rat Brain Using Liquid Chromatography–Mass Spectrometry

The brain is a lipid-rich organ containing complex polar lipids including phospholipids (PLs) and sphingolipids. These lipids are involved in the structure and function of cell membranes in the brain. We developed a fast and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify five different classes of PLs [Choline glycerophospholipid (consists of phosphatidyl choline and plasmenyl choline in these samples), ethanolamine glycerophospholipid (consist of phosphatidyl ethanolamine and plasmenyl ethanolamine in these samples), phosphatidyl serine, phosphatidyl inositol, and sphingomyelin] in the brain tissues of 80-day-old Wistar rats. The PLs were extracted from rat brain using chloroform/methanol/water. After separation using a hydrophilic high performance liquid chromatography column, PL-class-specific fragmentation (head group identification) with a tandem mass spectrometer in positive ion mode was utilized to measure changes in the relative concentration of the five PL classes. The advantage of this approach was its improved specificity over previously reported LC-MS methods. The method had good repeatability (coefficient of variation 3-9%, excluding phosphatidyl inositol) and recovery (92-103%) and compared well with more laborious traditional methods.

DHA enhances the noradrenaline release by SH-SY5Y cells

Polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and arachidonic acid (AA), are the main components of the phospholipids, in cerebral membranes. A dietary-induced cerebral DHA deficit results in altered behaviour and neurotransmission in rodents. To determine whether PUFA were acting on the neurotransmitter release machinery, we measured the release of [ 3H]-noradrenaline (NA) from SH-SY5Y neuroblastoma cells with modified PUFA membrane contents and from cells incubated with medium containing high DHA or AA. The membranes of cells incubated with 70 μM DHA for 3 days had 7.6-times more DHA in their ethanolamine glycerophospholipids, while the membranes of cells incubated with AA had 40% less. Incorporation of DHA enhanced basal [ 3H]-NA release (25%, p < 0.05), but not KCl-evoked [ 3H]-NA release. Brief incubation with DHA during vesicle mobilization also strongly increased [3H]-NA release. AA had no effect. The genes encoding for the calcium sensor synaptotagmin1, and for the two SNARE complex proteins syntaxin1A and synaptobrevin1 were not affected by PUFA incorporation, as indicated by assays for specific mRNAs and proteins. Thus both a high membrane DHA content and free DHA in the medium enhance the release of [ 3H]-NA from SH-SY5Y cells. This suggests that brain membrane DHA influences exocytosis, which then regulates neurotransmission.

In Vitro Growth Environment Produces Lipidomic and Electron Transport Chain Abnormalities in Mitochondria from Non-Tumorigenic Astrocytes and Brain Tumours

The mitochondrial lipidome influences ETC (electron transport chain) and cellular bioenergetic efficiency. Brain tumours are largely dependent on glycolysis for energy due to defects in mitochondria and oxidative phosphorylation. In the present study, we used shotgun lipidomics to compare the lipidome in highly purified mitochondria isolated from normal brain, from brain tumour tissue, from cultured tumour cells and from non-tumorigenic astrocytes. The tumours included the CT-2A astrocytoma and an EPEN (ependymoblastoma), both syngeneic with the C57BL/6J (B6) mouse strain. The mitochondrial lipidome in cultured CT-2A and EPEN tumour cells were compared with those in cultured astrocytes and in solid tumours grown in vivo. Major differences were found between normal tissue and tumour tissue and between in vivo and in vitro growth environments for the content or composition of ethanolamine glycerophospholipids, phosphatidylglycerol and cardiolipin. The mitochondrial lipid abnormalities in solid tumours and in cultured cells were associated with reductions in multiple ETC activities, especially Complex I. The in vitro growth environment produced lipid and ETC abnormalities in cultured non-tumorigenic astrocytes that were similar to those associated with tumorigenicity. It appears that the culture environment obscures the boundaries of the Crabtree and the Warburg effects. These results indicate that in vitro growth environments can produce abnormalities in mitochondrial lipids and ETC activities, thus contributing to a dependency on glycolysis for ATP production.

Calcium‐independent phospholipase A2‐mediated formation of 1,2‐diarachidonoyl‐glycerophosphoinositol in monocytes

Phagocytic cells exposed to exogenous arachidonic acid (AA) incorporate large quantities of this fatty acid into choline and ethanolamine glycerophospholipids, and into phosphatidylinositol (PtdIns). Utilizing liquid chromatography coupled to MS, we have characterized the incorporation of exogenous deuterated AA ([(2)H]AA) into specific PtdIns molecular species in human monocyte cells. A PtdIns species containing two exogenous [(2)H]AA molecules (1-[(2)H]AA-2-[(2)H]AA-glycero-3-phosphoinositol) was readily detected when human U937 monocyte-like cells and peripheral blood monocytes were exposed to [(2)H]AA concentrations as low as 160 nm to 1 mum. Bromoenol lactone, an inhibitor of Ca(2+)-independent phospholipase A(2) (iPLA(2)), diminished lyso-PtdIns levels, and almost completely inhibited the appearance of 1-[(2)H]AA-2-[(2)H]AA-glycero-3-phosphoinositol, suggesting the involvement of deacylation reactions in the synthesis of this phospholipid. De novo synthesis did not appear to be involved, as no other diarachidonoyl phospholipid or neutral lipid was detected under these conditions. Measurement of the metabolic fate of 1-[(2)H]AA-2-[(2)H]AA-glycero-3-phosphoinositol after pulse-labeling of the cells with [(2)H]AA showed a time-dependent, exponential decrease in the level of this phospholipid. These results identify 1-[(2)H]AA-2-[(2)H]AA-glycero-3-phosphoinositol as a novel, short-lived species for the initial incorporation of AA into the PtdIns class of cellular phospholipids in human monocytes.

An Improved Method for Separating Cardiolipin by HPLC

Herein we report an improved method to separate cardiolipin (Ptd(2)Gro) from tissue total lipid extracts using a biphasic solvent system combined with high performance liquid chromatography. This method uses a normal phase silica column and two mobile phases: mobile phase A that was n-hexane:2-propanol (3:2 by vol) and mobile phase B that was n-hexane:2-propanol:water (56.7:37.8:5.5 by vol). The initial solvent conditions were 95% A and 5% B, with a flow rate of 1.5 mL/min. The samples were from non-derivatized aliquots of liver, heart, or brain lipid extracts. The peak corresponding to Ptd(2)Gro appeared at 31 min, was well defined and did not overlap with neighboring peaks. The adjacent peak corresponded to ethanolamine glycerophospholipids and the remaining phospholipids were eluted in a single peak. The identity of the phospholipids separated by this method was verified by thin layer chromatography (TLC) and fatty acid analysis, which confirmed that the Ptd(2)Gro was well resolved from other phospholipids. This method is useful to separate and quantify Ptd(2)Gro from small tissue samples thereby avoiding the variability associated with TLC methods.

Brain Mitochondrial Lipid Abnormalities in Mice Susceptible to Spontaneous Gliomas

Alterations in mitochondrial function have long been considered a hallmark of cancer. We compared the lipidome and electron transport chain activities of non-synaptic brain mitochondria in two inbred mouse strains, the C57BL/6J (B6) and the VM/Dk (VM). The VM strain is unique in expressing a high incidence of spontaneous brain tumors (1.5%) that are mostly gliomas. The incidence of gliomas is about 210-fold greater in VM mice than in B6 mice. Using shotgun lipidomics, we found that the mitochondrial content of ethanolamine glycerophospholipid, phosphatidylserine, and ceramide was higher, whereas the content of total choline glycerophospholipid was lower in the VM mice than in B6 mice. Total cardiolipin content was similar in the VM and the B6 mice, but the distribution of cardiolipin molecular species differed markedly between the strains. B6 non-synaptic mitochondria contained 95 molecular species of cardiolipin that were symmetrically distributed over 7 major groups based on mass charge. In contrast, VM non-synaptic mitochondria contained only 42 molecular species that were distributed asymmetrically. The activities of Complex I, I/III, and II/III enzymes were lower, whereas the activity of complex IV was higher in the mitochondria of VM mice than in B6 mice. The high glioma incidence and alterations in electron transport chain activities in VM mice compared to B6 mice could be related to the unusual composition of mitochondrial lipids in the VM mouse brain.

The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations

The brain cannot synthesize n-6 or n-3 PUFAs de novo and requires their transport from the blood. Two models of brain fatty acid uptake have been proposed. One requires the passive diffusion of unesterified fatty acids through endothelial cells of the blood-brain barrier, and the other requires the uptake of lipoproteins via a lipoprotein receptor on the luminal membrane of endothelial cells. This study tested whether the low density lipoprotein receptor (LDLr) is necessary for maintaining brain PUFA concentrations. Because the cortex has a low basal expression of LDLr and the anterior brain stem has a relatively high expression, we analyzed these regions separately. LDLr knockout (LDLr(-/-)) and wild-type mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem (pons and medulla) were isolated for phospholipid fatty acid analyses. There were no differences in phosphatidylserine, phosphatidylinositol, ethanolamine, or choline glycerophospholipid esterified PUFA or saturated or monounsaturated fatty acid concentrations in the cortex or brain stem between LDLr(-/-) and wild-type mice. These findings demonstrate that the LDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFAs into the brain must exist.

Estradiol Favors the Formation of Eicosapentaenoic Acid (20:5n‐3) and n‐3 Docosapentaenoic Acid (22:5n‐3) from Alpha‐Linolenic Acid (18:3n‐3) in SH‐SY5Y Neuroblastoma Cells

Whether neurosteroids regulate the synthesis of long chain polyunsaturated fatty acids in brain cells is unknown. We examined the influence of 17-beta-estradiol (E2) on the capacity of SH-SY5Y cells supplemented with alpha-linolenic acid (ALA), to produce eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Cells were incubated for 24 or 72 h with ALA added alone or in combination with E2 (ALA + E2). Fatty acids were analyzed by gas chromatography of ethanolamine glycerophospholipids (EtnGpl) and phosphatidylcholine (PtdCho). Incubation for 24 h with ALA alone increased EPA and DPA in EtnGpl, by 330 and 430% compared to controls (P < 0.001) and DHA by only 10% (P < 0.05). Although DHA increased by 30% (P < 0.001) in ALA + E2-treated cells, the difference between the ALA and ALA + E2 treatments were not significant after 24 h (Anova-1, Fisher's test). After 72 h, EPA, DPA and DHA further increased in EtnGpl and PtdCho of cells supplemented with ALA or ALA + E2. Incubation for 72 h with ALA + E2 specifically increased EPA (+34% in EtnGpl, P < 0.001) and DPA (+15%, P < 0.001) compared to ALA alone. Thus, SH-SY5Y cells produced membrane EPA, DPA and DHA from supplemental ALA. The formation of DHA was limited, even in the presence of E2. E2 significantly favored EPA and DPA production in cells grown for 72 h. Enhanced synthesis of ALA-elongation products in neuroblastoma cells treated with E2 supports the hypothesis that neurosteroids could modulate the metabolism of PUFA.

Characterization of Acrolein-Glycerophosphoethanolamine Lipid Adducts Using Electrospray Mass Spectrometry

Acrolein is a toxic, highly reactive alpha,beta-unsaturated aldehyde. In the current study, the products of acrolein after reaction with glycerophosphoethanolamine (GPEtn) lipids have been characterized using electrospray tandem mass spectrometry. The major product formed involves the addition of two acrolein molecules to the primary amine of GPEtn lipids and subsequent aldol condensation to form 1,2-diradyl- sn-glycero-3-phosphoethanol-(3-formyl-4-hydroxy)piperidine (FHP) lipids. Upon sodium borohydride reduction, 1,2-diradyl- sn-glycero-3-phosphoethanol-(3-hydroxymethyl-4-hydroxy)piperidine (HMHP) lipids and 1,2-diradyl- sn-glycero-3-phosphoethanol-(3-hydroxymethyl-3,4-dehydro)piperidine (HMDP) lipids were selectively detected using electrospray tandem mass spectrometry by employing precursors of m/ z 256.1 and 238.1 scans, respectively. HMHP lipid and HMDP lipid molecular species were detected upon treatment of HL-60 cells with concentrations of acrolein as low as 10 microM. While the biological implications of these acrolein GPEtn adducts have yet to be established, these structural characterization studies reported herein reveal the facile formation of acrolein GPEtn lipid adducts in vitro, which could influence subsequent biochemical events within the cell.

Brain neutral lipids mass is increased in α‐synuclein gene‐ablated mice

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild-type and Snca-/- mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca-/- mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n-3) was decreased 7%, while 16 : 0 was increased 1.1-fold and 1.4-fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1-fold, 1.6-fold, and 1.4-fold, respectively in Snca-/- mice. In isolated myelin, cholesterol mass was also increased 1.3-fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.

α‐Synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unknown. In the present study, we examined the effect of Snca gene ablation on brain 22:6n-3 metabolism. We determined 22:6n-3 uptake and incorporation into brain phospholipids by infusing awake, wild-type and Snca-/- mice with [1-14C]22:6n-3 using steady-state kinetic modeling. In addition, because Snca modulates 20:4n-6-CoA formation, we assessed microsomal Acsl activity using 22:6n-3 as a substrate. Although Snca gene ablation does not affect brain 22:6n-3 uptake, brain 22:6n-3-CoA mass was elevated 1.5-fold in the absence of Snca. This is consistent with the 1.6- to 2.2-fold increase in the incorporation rate and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n-3-CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n-3, Kd = 1.0 +/- 0.5 micromol/L, it did not bind 22:6n-3-CoA. These effects of Snca gene deletion on 22:6n-3 brain metabolism are opposite to what we reported previously for brain 20:4n-6 metabolism and are likely compensatory for the decreased 20:4n-6 metabolism in brains of Snca-/- mice.

Lipid abnormalities in succinate semialdehyde dehydrogenase ( Aldh5a1− /− ) deficient mouse brain provide additional evidence for myelin alterations

Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1 − /− mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143–156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1 − /− brain. In comparison to wild-type littermates ( Aldh5a1 +/+ ), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1 − /− mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1 − /− brain tissue (one-tailed t test, p = 0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology.

Myocardial Lipidomics. Developments in myocardial nuclear lipidomics

The development of electrospray ionization mass spectrometry has been critical for the analyses of lipidomes from subcellular organelles. The myocardial nuclear lipidome likely has a key role in the molecular regulation of gene expression. In fact, recent studies have suggested that specific phospholipid classes bind and regulate specific transcription factors. The dynamic regulation of the myocardial nuclear lipidome may be critical in mediating long-term pathological responses to stresses such as ischemia, tachycardia, and hypertension. In this brief review, the preparation of myocardial nuclei is discussed, and the resulting nuclear lipidome from rat and rabbit are shown as examples. The rabbit myocardial nuclear lipidome contains relatively more plasmenylcholine/phosphatidylcholine molecular species in comparison to that ratio observed in the rat myocardial nuclear lipidome. The composition of the rat myocardial nuclear choline glycerophospholipid pool was relatively enriched with molecular species containing arachidonic acid and docosahexaenoic acid in comparison to that in the rabbit myocardial nuclear choline glycerophospholipid pool. While the ethanolamine glycerophospholipids of the rabbit myocardial nuclei are enriched with arachidonic acid and plasmalogens, the ethanolamine glycerophospholipid profile from rat myocardial nuclei show less plasmalogen and more species containing docosahexaenoic acid. Last, significant differences in the ethanolamine glycerophospholipid molecular species were observed in the rabbit heart lipidomes from the nucleus and the mitochondria. Quantitation of these lipid species in hearts subjected to pathophysiological stresses may provide important information on the role of the myocardial nuclear lipidome on long-term cardiac cell function.

Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A 2

We investigated the hydrolysis of the minor glycerophospholipids of human HDL 3, total HDL and LDL using human group IIA, V and X secretory phospholipases A 2 (sPLA 2s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA 2s, and to a lesser extent by group IIA sPLA 2, the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA 2s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA 2s, although the group V sPLA 2 attacked it more readily than group X sPLA 2 (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA 2 hydrolysis remained minimal at 10–15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA 2 at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA 2s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA 2 digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA 2s. Since these sPLA 2s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.

Cis-4,7,10,trans-13–22∶4 fatty acid distribution in phospholipids of pectinid species Aequipecten opercularis and Pecten maximus

The distribution of cis-4,7,10,trans-13-docosatetraenoic (c4,7,10,t13-22:4), a peculiar FA previously isolated in the glycerophospholipids of some pectinid bivalves, was investigated in glycerophospholipid classes and subclasses of separated organs (gills, mantle, gonads, and muscle) of the queen scallop Aequipecten opercularis and the king scallop Pecten maximus. Plasmalogen (Pls) and diacyl + alkyl (Ptd) forms of serine, ethanolamine, and choline glycerophospholipids were isolated by HPLC and their FA compositions analyzed by GC-FID. PIs and Ptd forms of serine glycerophospholipids (PlsSer and PtdSer), and to a lesser extend the Pls form of ethanolamine glycerophospholipids (PlsEtn), were found to be specifically enriched with c4,7,10,t13-22:4. This specificity was found to decrease in the tested organs in the following order: gills, mantle, gonad, and muscle. In gills, c4,7,10,t13-22:4 was shown to be the main unsaturated FA of serine glycerophospholipids in both Pls and Ptd forms (23.8 and 19.4 mol%, respectively, for A. opercularis, and 21.0 and 26.2 mol% for P. maximus). These results represent the first comprehensive report on the FA composition of plasmalogen serine subclass isolated from pectinid bivalves. The specific association of the PlsSer with the c4,7,10,t13-22:4 for the two pectinid species can be paralleled to the specific association of the PlsSer with the non-methylene interrupted (NMI) FA and 20:1 (n-11) observed in mussels, clams, and oysters (Kraffe, E., Soudant, P., and Marty, Y. (2004) Fatty Acids of Serine, Ethanolamine and Choline Plasmalogens in Some Marine Bivalves, Lipids 39, 59-66.) This, led us to hypothesize a similar functional significance for c4,7,10,t13-22:4, NMI FA, and 20:1 (n-11) associated with PlsSer subclass of bivalves.

Analysis of phospholipid species in rat peritoneal surface layer by liquid chromatography/electrospray ionization ion-trap mass spectrometry

The main phospholipids in rat peritoneal surface layer were analyzed by normal-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion-trap mass spectrometry (MS). By using a silica gel column and a gradient of hexane/isopropanol/water as mobile phase containing 5 mmol/L ammonium formate as modifiers, a baseline separation of glycerophosphoehtanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and lyso-phosphatidylcholine (LPC) was obtained and more than 90 phospholipid constituents in rat peritoneal surface were identified and determined by on-line ion-trap MS detection. The major ethanolamine glycerophospholipids in rat peritoneal surfaces were plasmalogens that were highly enriched in polyunsaturated fatty acids at the sn-2 position. In addition, the fragmentation patterns for each phospholipid class by the ion-trap MS were discussed.

Selective remodeling of cardiolipin fatty acids in the aged rat heart

BackgroundThe heart is rich in cardiolipin, a phospholipid acylated in four sites, predominately with linoleic acid. Whether or not aging alters the composition of cardiolipin acyl chains is controversial. We therefore measured the fatty acid concentration of cardiolipin in hearts of 4, 12 and 24 month old rats that consumed one diet, adequate in fatty acids for the duration of their life.ResultsThe concentration (nmol/g) of linoleic acid was decreased in 24 month old rats (3965 ± 617, mean ± SD) vs 4 month old rats (5525 ± 656), while the concentrations of arachidonic and docosahexaenoic acid were increased in 24 month old rats (79 ± 9 vs 178 ± 27 and 104 ± 16 vs 307 ± 68 for arachidonic and docosahexaenoic acids, 4 months vs 24 months, respectively). Similar changes were not observed in ethanolamine glycerophospholipids or plasma unesterified fatty acids, suggesting specificity of these effects to cardiolipin.ConclusionThese results demonstrate that cardiolipin remodeling occurs with aging, specifically an increase in highly unsaturated fatty acids.

Myeloperoxidase-derived 2-chlorohexadecanal forms Schiff bases with primary amines of ethanolamine glycerophospholipids and lysine

Numerous studies have suggested relationships between myeloperoxidase, inflammation, and atherosclerosis. MPO-derived reactive chlorinating species (RCS) attack membrane plasmalogens releasing α-chloro-fatty aldehydes (α-Cl-FALDs) including 2-chlorohexadecanal (2-ClHDA). The molecular targets of α-Cl-FALDs are not known. The current study demonstrates 2-ClHDA adducts with ethanolamine glycerophospholipids and Fmoc-lysine. Utilizing electrospray ionization mass spectrometry, chlorinated adducts were observed that are apparent Schiff base adducts. Reduction of these Schiff base adducts with sodium cyanoborohydride resulted in a novel, stable adduct produced by the elimination of HCl. NMR further confirmed this structure. 2-ClHDA adducts with ethanolamine glycerophospholipids were also substrates for phospholipase D (PLD). The hydrolysis products were derivatized to pentafluorobenzoyl esters, and further structurally confirmed by GC–MS. Multiple molecular species of 2-ClHDA- N-modified ethanolamine glycerophospholipids were observed in endothelial cells treated with 2-ClHDA. These results show novel Schiff base adducts of α-Cl-FALDs with primary amines, which may represent an important fate of α-Cl-FALDs.