Choline Glycerophospholipids Research Articles

Disturbed brain arachidonic acid metabolism in HIV‐1 transgenic rats

Human immunodeficiency virus (HIV) is associated with neuroinflammation and related neuropathological co-morbidities, such as HIV-associated dementia and psychosis, following viral entry into the brain. The entry of viral-bearing HIV monocytes into the brain is associated with microglial activation, neuroinflammation and increased arachidonic acid (AA) signalling (Basselin M et al., J Cereb Blood Flow Metab., 2010), which is elevated in pathological conditions of excitotoxicity and inflammation. We hypothesized that AA turnover due to metabolic loss (of AA) in the brain, would be elevated in HIV-transgenic rats (HIV-Tg), a non-infectious HIV-1 model associated with neuroinflammation and behavioural abnormalities after 5 months of age. Brian AA turnover and fatty acid concentrations were measured after [1-(14)C]AA infusion to 7–9 months old, unanesthetized rats. Brain AA turnover due to metabolic loss increased by ~2-fold, and concentrations decreased by 14–18% in ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylinositol and phosphatidylserine of HIV-Tg rats relative to controls. The results suggest upregulated AA metabolism in HIV-Tg rats, in which behavioural abnormalities, neurologic changes and neuroinflammation have been reported. This work was supported by the National Institute on Aging Intramural Program. Grant Funding Source: Intramural Research Funding from the National Institute on Aging

Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm–oocyte interactions.

Markers of Monocyte Activation Revealed by Lipidomic Profiling of Arachidonic Acid-Containing Phospholipids

Stimulated human monocytes undergo an intense trafficking of arachidonic acid (AA) among glycerophospholipidclasses. Using HPLC coupled to electrospray ionization mass spectrometry, we have characterized changes in the levels of AA-containing phospholipid species in human monocytes. In resting cells, AA was found esterified into various molecular species of phosphatidylinositol (PI), choline glycerophospholipids (PCs), and ethanolamine glycerophospholipids (PEs). All major AA-containing PC and PI molecular species decreased in zymosan-stimulated cells; however, no PE molecular species was found to decrease. In contrast, the levels of three AA-containing species increased in zymosan-activated cells compared with resting cells: 1,2-diarachidonyl-glycero-3-phosphoinositol [PI(20:4/20:4)]; 1,2-diarachidonyl-glycero-3-phosphocholine [PC(20:4/20:4)]; and 1-palmitoleoyl-2-arachidonyl-glycero-3-phosphoethanolamine [PE(16:1/20:4)]. PI(20:4/20:4) and PC(20:4/20:4), but not PE(16:1/20:4), also significantly increased when platelet-activating factor or PMA were used instead of zymosan to stimulate the monocytes. Analysis of the pathways involved in the synthesis of these three lipids suggest that PI(20:4/20:4) and PC(20:4/20:4) were produced in a deacylation/reacylation pathway via acyl-CoA synthetase-dependent reactions, whereas PE(16:1/20:4) was generated via a CoA-independent transacylation reaction. Collectively, our results define the increases in PI(20:4/20:4) and PC(20:4/20:4) as lipid metabolic markers of human monocyte activation and establish lipidomics as a powerful tool for cell typing under various experimental conditions.

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.

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.

Systematic analysis of choline-containing phospholipids using multi-dimensional mass spectrometry-based shotgun lipidomics

Herein, a systematic study on the identification and quantitation of choline-containing phospholipid molecular species, including choline glycerophospholipid (PC), lysoPC, and sphingomyelin (SM), is described using multi-dimensional mass spectrometry-based shotgun lipidomics after intrasource separation (MDMS-SL). Current methods for analysis of choline-containing lipids were improved through multiple modifications leading to: (1) identification of constituents present in the PC and SM classes, subclasses of PC, and individual molecular species using MDMS-SL analysis in the positive-ion mode; (2) identification of the fatty acyl constituents and their regiospecificity in diacyl PC molecular species through the neutral loss of trimethylamine plus fatty acids; (3) direct identification of the alkenyl chains of plasmenylcholine species using precursor ion scans of the fragment ions carrying the alkenyl chains; (4) elimination of the effects of polyunsaturation on the quantitation of PC species by multiple ratiometric comparisons; (5) accurate identification and quantitation of lysoPC molecular species including regioisomers by diagnostic fragment ions; and (6) accurate identification and quantitation of SM molecular species by neutral loss scans of phosphocholine plus methyl aldehyde which is specific to SM molecular species. With these enhancements, the application of MDMS-SL for the analyses of choline-containing phospholipid molecular species in biomedical research has been extended to a much larger number of molecular species with greater quantitative accuracy and an increased depth of structural information.

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.

Scavenger Receptor A and CD36 Are Implicated in Mediating Platelet Activation Induced by Oxidized Low- Density Lipoproteins

See ATVB 2007;2476–2483. CD36, previously known as GPIIIb or GPIV, is an 88-kDa membrane glycoprotein that was isolated from blood platelets soon after being identified on monocytes by monoclonal antibody OKM5. Cloning the CD36 gene showed this cell adhesion molecule to be a member of the scavenger receptor class B family. Moreover, it was observed that CD36 has the capacity to bind oxidized low-density lipoproteins (oxLDL) but not acetylated lipoproteins. Scavenger receptor class A (SRA),1 also expressed on platelets and monocytes/macrophages, is a trimer of 77 kDa, that binds to acetylated and oxidized lipoproteins. SRA is closely associated to macrophage CD36 in initiating atherosclerotic lesions, in some studies but not all.2,3,4 Very little was known, up to now, on the role played by oxLDL in activating platelets via CD36 and SRA and promoting a prothrombotic phenotype. The study, in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , by Suzanne Korporaal and coworkers,5 shows very elegantly that oxLDL, in tandem with CD36 and SRA, is activating platelets and potentially increasing the risk of thrombosis. This investigation brings some exciting data on a novel platelet-activating pathway triggered by partially oxidized (30% to 60%) LDL and mediated by the combined activity of CD36 and …

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Abstract 522: Specific Oxidized Phospholipids Modulate Platelet Reactivity In Vivo During Hyperlipidemia Via Cd36: A Mechanism Linking Hyperlipidemia, Oxidant Stress And A Pro-thrombotic Phenotype

Enhanced platelet reactivity is critical to the pathophysiology of occlusive arterial thrombotic disease. Despite the strong clinical associations between hyperlipidemia, a major risk factor for atherosclerosis, and a pro-thrombotic phenotype, the mechanisms responsible for enhanced platelet reactivity during hyperlipidemia remain unknown. Pro-atherosclerotic lipid abnormalities such as hypercholesterolemia are associated with both enhanced oxidant stress and generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet surface glycoprotein. Using multiple murine in vivo thrombosis models we now demonstrate that hyperlipidemic atherosclerosis prone mice (apolipoprotein E-deficient or LDL receptor-deficient) form occlusive intravascular thrombi faster than wildtype mice, and that genetic deletion of CD36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and accompanying pro-thrombotic phenotype. Structurally defined oxidized choline glycerophospholipid molecular species that serve as endogenous high affinity ligands for CD36 are shown to : be markedly increased in plasma of hyperlipidemic mice; be elevated in plasma of subjects with low HDL levels; and to promote platelet activation and alpha-granule release via CD36 at pathophysiological levels. These studies thus demonstrate that platelet CD36 interactions with specific endogenous oxidized lipids play a heretofore unrecognized role in the well-known clinical associations between hyperlipidemia, oxidant stress and a prothrombotic phenotype.

Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype

Dyslipidemia is associated with a prothrombotic phenotype; however, the mechanisms responsible for enhanced platelet reactivity remain unclear. Proatherosclerotic lipid abnormalities are associated with both enhanced oxidant stress and the generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet glycoprotein. Using multiple mouse in vivo thrombosis models, we now demonstrate that genetic deletion of Cd36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and the accompanying prothrombotic phenotype. Structurally defined oxidized choline glycerophospholipids that serve as high-affinity ligands for CD36 were at markedly increased levels in the plasma of hyperlipidemic mice and in the plasma of humans with low HDL levels, were able to bind platelets via CD36 and, at pathophysiological levels, promoted platelet activation via CD36. Thus, interactions of platelet CD36 with specific endogenous oxidized lipids play a crucial role in the well-known clinical associations between dyslipidemia, oxidant stress and a prothrombotic phenotype.

Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

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.

Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPC(CD36)), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517-38523). A high affinity CD36 recognition motif was defined within oxPC(CD36), an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPC(CD36), a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPC(CD36) precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain ( sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC–chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC–ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC–ClOH.

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.

Light-induced Oxidation of Photoreceptor Outer Segment Phospholipids Generates Ligands for CD36-mediated Phagocytosis by Retinal Pigment Epithelium: A POTENTIAL MECHANISM FOR MODULATING OUTER SEGMENT PHAGOCYTOSIS UNDER OXIDANT STRESS CONDITIONS

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.

α-Synuclein Gene Deletion Decreases Brain Palmitate Uptake and Alters the Palmitate Metabolism in the Absence of α-Synuclein Palmitate Binding

Alpha-synuclein is an abundant protein in the central nervous system that is associated with a number of neurodegenerative disorders, including Parkinson's disease. Its physiological function is poorly understood, although recently it was proposed to function as a fatty acid binding protein. To better define a role for alpha-synuclein in brain fatty acid uptake and metabolism, we infused awake, wild-type, or alpha-synuclein gene-ablated mice with [1-(14)C]palmitic acid (16:0) and assessed fatty acid uptake and turnover kinetics in brain phospholipids. Alpha-synuclein deficiency decreased brain 16:0 uptake 35% and reduced its targeting to the organic fraction. The incorporation coefficient for 16:0 entering the brain acyl-CoA pool was significantly decreased 36% in alpha-synuclein gene-ablated mice. Because incorporation coefficients alone are not predictive of fatty acid turnover in individual phospholipid classes, we calculated kinetic values for 16:0 entering brain phospholipid pools. Alpha-synuclein deficiency decreased the incorporation rate and fractional turnover of 16:0 in a number of phospholipid classes, but also increased the incorporation rate and fractional turnover of 16:0 in the choline glycerophospholipids. No differences in incorporation rate or turnover were observed in liver phospholipids, confirming that these changes in lipid metabolism were brain specific. Using titration microcalorimetry, we observed no binding of 16:0 or oleic acid to alpha-synuclein in vitro. Thus, alpha-synuclein has effects on 16:0 uptake and metabolism similar to those of an FABP, but unlike FABP, it does not directly bind 16:0; hence, the mechanism underlying these effects is different from that of a classical FABP.