Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR
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Keywords:
Isotopologue
Isotopomers
Glycerophospholipids
Glycerophospholipids
Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained. Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained. ERRATUMJournal of Lipid ResearchVol. 55Issue 11PreviewThe authors of "Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells" (J. Lipid Res. 2014. 55: 799–807) have advised that they inadvertently introduced errors into their Table 1. The corrected text is shown in bold in the shaded area in the table below. Full-Text PDF Open Access One of the major components of cellular membranes is a class of molecules known as glycerophospholipids, which are synthesized from glycerol-3-phosphate (G3P) in a de novo pathway that initially produces phosphatidic acid (PA) and diacylglycerol (DAG) or cytidine diphosphate-DAG (CDP-DAG) (1Kennedy E.P. The synthesis of cytidine diphosphate choline, cytidine diphosphate ethanolamine, and related compounds.J. Biol. Chem. 1956; 222: 185-191Abstract Full Text PDF PubMed Google Scholar, 2Kennedy E.P. The biological synthesis of phospholipids.Can. J. Biochem. Physiol. 1956; 34: 334-348Crossref PubMed Google Scholar, 3Holub B.J. Kuksis A. Metabolism of molecular species of diacylglycerophospholipids.Adv. Lipid Res. 1978; 16: 1-125Crossref PubMed Google Scholar). Via the de novo pathway, various types of glycerophospholipids with different polar heads at the sn-3 position in the glycerol backbone, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL) are generated (4van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4135) Google Scholar, 5Yamashita A. Hayashi Y. Nemoto-Sasaki Y. Ito M. Oka S. Tanikawa T. Waku K. Sugiura T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms.Prog. Lipid Res. 2014; 53: 18-81Crossref PubMed Scopus (148) Google Scholar). Subsequently, glycerophospholipid acyl chains are remodeled by the orchestrated reactions of phospholipase As (PLAs), acyl-CoA synthases, transacylases, and lysophospholipid acyltransferases (LPLATs) (5Yamashita A. Hayashi Y. Nemoto-Sasaki Y. Ito M. Oka S. Tanikawa T. Waku K. Sugiura T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms.Prog. Lipid Res. 2014; 53: 18-81Crossref PubMed Scopus (148) Google Scholar, 6Murakami M. Taketomi Y. Miki Y. Sato H. Hirabayashi T. Yamamoto K. Recent progress in phospholipase A(2) research: from cells to animals to humans.Prog. Lipid Res. 2011; 50: 152-192Crossref PubMed Scopus (356) Google Scholar, 7Kita Y. Ohto T. Uozumi N. Shimizu T. Biochemical properties and pathophysiological roles of cytosolic phospholipase A2s.Biochim. Biophys. 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Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells.J. Biochem. 1997; 122: 1-16Crossref PubMed Google Scholar). Thus far, investigations of glycerophospholipid remodeling have mainly focused on PLAs, especially in the production of lipid mediators (6Murakami M. Taketomi Y. Miki Y. Sato H. Hirabayashi T. Yamamoto K. Recent progress in phospholipase A(2) research: from cells to animals to humans.Prog. Lipid Res. 2011; 50: 152-192Crossref PubMed Scopus (356) Google Scholar, 7Kita Y. Ohto T. Uozumi N. Shimizu T. Biochemical properties and pathophysiological roles of cytosolic phospholipase A2s.Biochim. Biophys. Acta. 2006; 1761: 1317-1322Crossref PubMed Scopus (96) Google Scholar, 8Shimizu T. Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 123-150Crossref PubMed Scopus (388) Google Scholar). However, in recent years, various LPLATs have been identified from the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) and membrane bound O-acyltransferase (MBOAT) families (Table 1). Although studies with tissue homogenates initially suggested that each LPLAT recognizes a specific substrate, isolated LPLATs have shown promiscuous substrate specificities (5Yamashita A. Hayashi Y. Nemoto-Sasaki Y. Ito M. Oka S. Tanikawa T. Waku K. Sugiura T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms.Prog. Lipid Res. 2014; 53: 18-81Crossref PubMed Scopus (148) Google Scholar, 9Shindou H. Hishikawa D. Harayama T. Yuki K. Shimizu T. Recent progress on acyl CoA: lysophospholipid acyltransferase research.J. Lipid Res. 2009; 50: S46-S51Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 12Shindou H. Hishikawa D. Harayama T. Eto M. Shimizu T. Generation of membrane diversity by lysophospholipid acyltransferases.J. Biochem. 2013; 154: 21-28Crossref PubMed Scopus (81) Google Scholar). Because the acyl composition of membrane glycerophospholipids is known to affect not only the production of lipid mediators but also membrane properties, characterizing these LPLATs will reveal the biological importance of membrane glycerophospholipid diversity.TABLE 1.Summary of characteristics of LPLATsSubstrate In VitroNameOther NamesLysophospholipidAcyl-CoAExpressionPhenotypes of KO, Knockdown, and Mutations In VivoReferencesLPAAT1aAGPAT family member.AGPAT1, LPAATαLPA—Ubiquitous—(117West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. et al.Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (111) Google Scholar–119Kume K. Shimizu T. cDNA cloning and expression of murine 1-acyl-sn-glycerol-3-phosphate acyltransferase.Biochem. Biophys. Res. Commun. 1997; 237: 663-666Crossref PubMed Scopus (49) Google Scholar)LPAAT2aAGPAT family member.AGPAT2, LPAATβLPA, LPI—Adipose, liver, pancreas, heartLipodystrophy, diabetes(117West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. et al.Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.DNA Cell Biol. 1997; 16: 691-701Crossref PubMed Scopus (111) Google Scholar, 120Eberhardt C. Gray P.W. Tjoelker L.W. Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3.J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar–123Cortés V.A. Curtis D.E. Sukumaran S. Shao X. Parameswara V. Rashid S. Smith A.R. Ren J. Esser V. Hammer R.E. et al.Molecular mechanisms of hepatic steatosis and insulin resistance in the AGPAT2-deficient mouse model of congenital generalized lipodystrophy.Cell Metab. 2009; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar)LPAAT3aAGPAT family member.AGPAT3, LPAATγLPA, LPG, LPC, LPE, lyso-PAFPUFA-CoATestis, adipose, liver, kidney—(20Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. Lipid Res. 2009; 50: 860-869Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 122Vergnes L. Beigneux A.P. Davis R. Watkins S.M. Young S.G. 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Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production.J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar–44Bridges J.P. Ikegami M. Brilli L.L. Chen X. Mason R.J. Shannon J.M. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice.J. Clin. Invest. 2010; 120: 1736-1748Crossref PubMed Scopus (98) Google Scholar, 46Friedman J.S. Chang B. Krauth D.S. Lopez I. Waseem N.H. Hurd R.E. Feathers K.L. Branham K.E. Shaw M. Thomas G.E. et al.Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice.Proc. Natl. Acad. Sci. USA. 2010; 107: 15523-15528Crossref PubMed Scopus (47) Google Scholar, 125Soupene E. Fyrst H. Kuypers F.A. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.Proc. Natl. Acad. 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USA. 2008; 105: 88-93Crossref PubMed Scopus (77) Google Scholar)LPCAT3bMBOAT family member.MBOAT5LPC, LPEPUFA-CoATestis, liver, kidneyHepatic inflammation in ob/ob mouse(17Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (196) Google Scholar, 18Zhao Y. Chen Y.Q. Bonacci T.M. Bredt D.S. Li S. Bensch W.R. Moller D.E. Kowala M. Konrad R.J. Cao G. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.J. Biol. Chem. 2008; 283: 8258-8265Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 26Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 29Rong X. Albert C.J. Hong C. Duerr M.A. Chamberlain B.T. Tarling E.J. Ito A. Gao J. Wang B. Edwards P.A. et al.LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition.Cell Metab. 2013; 18: 685-697Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 127Matsuda S. Inoue T. Lee H.C. Kono N. Tanaka F. Gengyo-Ando K. Mitani S. Arai H. Member of the membrane-bound O-acyltransferase (MBOAT) family encodes a lysophospholipid acyltransferase with broad substrate specificity.Genes Cells. 2008; 13: 879-888Crossref PubMed Scopus (55) Google Scholar)LPCAT4bMBOAT family member.MBOAT2LPE, LPSOleoyl-CoATestis, epididymis, ovary, brain—(17Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (196) Google Scholar, 26Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar)LPEAT1bMBOAT family member.MBOAT1LPEOleoyl-CoATestis, epididymis, ovary, brainBrachydactyly-syndactyly syndrome(17Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (196) Google Scholar, 26Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar)LPEAT2aAGPAT family member.AGPAT7, Aytl3, LPCAT4LPI—Brain—(128Cao J. Shan D. Revett T. Li D. Wu L. Liu W. Tobin J.F. Gimeno R.E. Molecular identification of a novel mammalian brain isoform of acyl-CoA:lysophospholipid acyltransferase with prominent ethanolamine lysophospholipid acylating activity, LPEAT2.J. Biol. Chem. 2008; 283: 19049-19057Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar)LPIAT1bMBOAT family member.MBOAT7, MBOA7LPIPUFA-CoAUbiquitousPostnatal lethal, atrophy of the cerebral cortex and hippocampus, altered fatty acid composition of PI and PIPs(19Lee H.C. Inoue T. Imae R. Kono N. Shirae S. Matsuda S. Gengyo-Ando K. Mitani S. Arai H. Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol.Mol. Biol. Cell. 2008; 19: 1174-1184Crossref PubMed Scopus (100) Google Scholar, 20Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. 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Open table in a new tab Gene names, families, substrates preferences, mRNA expression patterns, and in vivo functions of LPLATs are summarized. Please note that there are several inconsistent reports about the enzymatic substrates in vitro. In mammalian cells, glycerophospholipid composition differs among cell types, organelles, and inner/outer membranes, and these differences are known to play important roles in various cellular functions including signal transduction, vesicle trafficking, and membrane fluidity (4van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4135) Google Scholar). Recently, several molecules involved in phospholipid transport between membranes and in phospholipid scrambling in plasma membranes have been identified. These factors are also important for constructing the specific composition of local membranes. In this review, we summarize and discuss the biological importance of the variety of membrane glycerophospholipids generated via glycerophospholipid remodeling by LPLATs. Glycerophospholipid remodeling by the concerted action of PLAs and LPLATs is important for the production of PUFA-containing glycerophospholipids (8Shimizu T. Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 123-150Crossref PubMed Scopus (388) Google Scholar). Glycerophospholipids containing PUFAs, such as arachidonic acid, linoleic acid, EPA, and DHA, are known as major sources of fatty acid-derived lipid mediators and endocannabinoids (8Shimizu T. Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 123-150Crossref PubMed Scopus (388) Google Scholar, 13Serhan C.N. Chiang N. Van Dyke T.E. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators.Nat. Rev. Immunol. 2008; 8: 349-361Crossref PubMed Scopus (2094) Google Scholar, 14Vangaveti V. Baune B.T. Kennedy R.L. Hydroxyoctadecadienoic acids: novel regulators of macrophage differentiation and atherogenesis.Ther. Adv. Endocrinol. Metab. 2010; 1: 51-60Crossref PubMed Scopus (107) Google Scholar, 15Piomelli D. Sasso O. Peripheral gating of pain signals by endogenous lipid mediators.Nat. Neurosci. 2014; 17: 164-174Crossref PubMed Scopus (158) Google Scholar). Although numerous studies have shown the importance of PLAs in producing lipid mediators, the involvement of LPLATs in lipid mediator production is poorly understood (6Murakami M. Taketomi Y. Miki Y. Sato H. Hirabayashi T. Yamamoto K. Recent progress in phospholipase A(2) research: from cells to animals to humans.Prog. Lipid Res. 2011; 50: 152-192Crossref PubMed Scopus (356) Google Scholar, 7Kita Y. Ohto T. Uozumi N. Shimizu T. Biochemical properties and pathophysiological roles of cytosolic phospholipase A2s.Biochim. Biophys. Acta. 2006; 1761: 1317-1322Crossref PubMed Scopus (96) Google Scholar). At present, lyso-PC (LPC) acyltransferase (LPCAT)2, LPCAT3, lyso-PI (LPI) acyltransferase (LPIAT)1, and lyso-PA (LPA) acyltransferase (LPAAT)3 are reported to incorporate PUFAs into lysophospholipids with different acceptor preferences (16Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase.J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 17Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (196) Google Scholar, 18Zhao Y. Chen Y.Q. Bonacci T.M. Bredt D.S. Li S. Bensch W.R. Moller D.E. Kowala M. Konrad R.J. Cao G. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.J. Biol. Chem. 2008; 283: 8258-8265Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 19Lee H.C. Inoue T. Imae R. Kono N. Shirae S. Matsuda S. Gengyo-Ando K. Mitani S. Arai H. Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selective incorporation of polyunsaturated fatty acids into phosphatidylinositol.Mol. Biol. Cell. 2008; 19: 1174-1184Crossref PubMed Scopus (100) Google Scholar, 20Yuki K. Shindou H. Hishikawa D. Shimizu T. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.J. Lipid Res. 2009; 50: 860-869Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). LPCAT3 is ubiquitously expressed, especially in liver, testis, kidney, pancreas, and adipose tissue (17Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.Proc. Natl. Acad. Sci. USA. 2008; 105: 2830-2835Crossref PubMed Scopus (196) Google Scholar, 18Zhao Y. Chen Y.Q. Bonacci T.M. Bredt D.S. Li S. Bensch W.R. Moller D.E. Kowala M. Konrad R.J. Cao G. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.J. Biol. Chem. 2008; 283: 8258-8265Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Expression of LPCAT3 mRNA is controlled by PPARα and liver X receptors, and is induced during adipogenesis (21Demeure O. Lecerf F. Duby C. Desert C. Ducheix S. Guillou H. Lagarrigue S. Regulation of LPCAT3 by LXR.Gene. 2011; 470: 7-11Crossref PubMed Scopus (0) Google Scholar, 22Eto M. Shindou H. Koeberle A. Harayama T. Yanagida K. Shimizu T. Lysophosphatidylcholine acyltransferase 3 is the key enzyme for incorporating arachidonic acid into glycerophospholipids during adipocyte differentiation.Int. J. Mol. Sci. 2012; 13: 16267-16280Crossref PubMed Scopus (23) Google Scholar). Knockdown of LPCAT3 by siRNA reduces arachidonic acid incorporation into PC and production of eicosanoids (23Ishibashi M. Varin A. Filomenko R. Lopez T. Athias A. Gambert P. Blache D. Thomas C. Gautier T. Lagrost L. et al.Liver X receptor regulates arachidonic acid distribution and eicosanoid release in human macrophages: a key role for lysophosphatidylcholine acyltransferase 3.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1171-1179Crossref PubMed Scopus (44) Google Scholar). Similar results were obtained from the treatment of thimerosal, a LPLAT inhibitor, and triacsin C, an acyl-CoA synthetase inhibitor (24Goppelt-Struebe M. Koerner C.F. Hausmann G. Gemsa D. Resch K. Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids.Prostaglandins. 1986; 32: 373-385Crossref PubMed Google Scholar, 25Kaever V. Goppelt-Strube M. Resch K. Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal.Prostaglandins. 1988; 35: 885-902Crossref PubMed Scopus (0) Google Scholar, 26Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 27Hartman E.J. Omura S. Laposata M. Triacsin C: a differential inhibitor of arachidonoyl-CoA synthetase and nonspecific long chain acyl-CoA synthetase.Prostaglandins. 1989; 37: 655-671Crossref PubMed Scopus (40) Google Scholar, 28Kuwata H. Yoshimura M. Sasaki Y. Yoda E. Nakatani Y. Kudo I. Hara S. Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts.Biochim. Biophys. Acta. 2014; 1841: 44-53Crossref PubMed Scopus (31) Google Scholar). These reports suggested that the control of arachidonic acid pools in membrane glycerophospholipids is important for the eicosanoid production. Furthermore, it has been reported that induction of LPCAT3 ameliorates saturated free fatty acid-induced endoplasmic reticulum (ER) stress in vitro (29Rong X. Albert C.J. Hong C. Duerr M.A. Chamberlain B.T. Tarling E.J. Ito A. Gao J. Wang B. Edwards P.A. et al.LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition.Cell Metab. 2013; 18: 685-697Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Using liver-specific LPCAT3 overexpression and knockdown mice, the study demonstrated that LPCAT3 regulates hepatic inflammatory cytokine levels and inflammation. Although the exact mechanism is unclear, the authors s
Glycerophospholipids
Glycerophospholipids
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Acyltransferases
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The METLIN metabolite database has become one of the most widely used resources in metabolomics for making metabolite identifications. However, METLIN is not designed to identify metabolites that have been isotopically labeled. As a result, unbiasedly tracking the transformation of labeled metabolites with isotope-based metabolomics is a challenge. Here, we introduce a new database, called isoMETLIN (http://isometlin.scripps.edu/), that has been developed specifically to identify metabolites incorporating isotopic labels. isoMETLIN enables users to search all computed isotopologues derived from METLIN on the basis of mass-to-charge values and specified isotopes of interest, such as (13)C or (15)N. Additionally, isoMETLIN contains experimental MS/MS data on hundreds of isotopomers. These data assist in localizing the position of isotopic labels within a metabolite. From these experimental MS/MS isotopomer spectra, precursor atoms can be mapped to fragments. The MS/MS spectra of additional isotopomers can then be computationally generated and included within isoMETLIN. Given that isobaric isotopomers cannot be separated chromatographically or by mass but are likely to occur simultaneously in a biological system, we have also implemented a spectral-mixing function in isoMETLIN. This functionality allows users to combine MS/MS spectra from various isotopomers in different ratios to obtain a theoretical MS/MS spectrum that matches the MS/MS spectrum from a biological sample. Thus, by searching MS and MS/MS experimental data, isoMETLIN facilitates the identification of isotopologues as well as isotopomers from biological samples and provides a platform to drive the next generation of isotope-based metabolomic studies.
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Kinetic isotope effect
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Glycerophospholipids
Glycerophospholipids
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A large scale profiling and analysis of glycerophospholipid species in macrophages has facilitated the identification of several rare and atypical glycerophospholipid species. By using liquid chromatography tandem mass spectrometry and comparison of the elution and fragmentation properties of the rare lipids to synthetic standards, we were able to identify an array of ether-linked phosphatidylinositols (PIs), phosphatidic acids, phosphatidylserines (PSs), very long chain phosphatidylethanolamines (PEs), and phosphatidylcholines (PCs) as well as phosphatidylthreonines (PTs) and a wide collection of odd carbon fatty acid-containing phospholipids in macrophages. A comprehensive qualitative analysis of glycerophospholipids from different macrophage cells was conducted. During the phospholipid profiling of the macrophage-like RAW 264.7 cells, we identified dozens of rare or previously uncharacterized phospholipids, including ether-linked PIs, PSs, and glycerophosphatidic acids, PTs, and PCs and PTs containing very long polyunsaturated fatty acids. Additionally, large numbers of phospholipids containing at least one odd carbon fatty acid were identified. Using the same methodology, we also identified many of the same species of glycerophospholipids in resident peritoneal macrophages, foam cells, and murine bone marrow derived macrophages. A large scale profiling and analysis of glycerophospholipid species in macrophages has facilitated the identification of several rare and atypical glycerophospholipid species. By using liquid chromatography tandem mass spectrometry and comparison of the elution and fragmentation properties of the rare lipids to synthetic standards, we were able to identify an array of ether-linked phosphatidylinositols (PIs), phosphatidic acids, phosphatidylserines (PSs), very long chain phosphatidylethanolamines (PEs), and phosphatidylcholines (PCs) as well as phosphatidylthreonines (PTs) and a wide collection of odd carbon fatty acid-containing phospholipids in macrophages. A comprehensive qualitative analysis of glycerophospholipids from different macrophage cells was conducted. During the phospholipid profiling of the macrophage-like RAW 264.7 cells, we identified dozens of rare or previously uncharacterized phospholipids, including ether-linked PIs, PSs, and glycerophosphatidic acids, PTs, and PCs and PTs containing very long polyunsaturated fatty acids. Additionally, large numbers of phospholipids containing at least one odd carbon fatty acid were identified. Using the same methodology, we also identified many of the same species of glycerophospholipids in resident peritoneal macrophages, foam cells, and murine bone marrow derived macrophages. murine bone marrow derived macrophages isopropyl alcohol Lipid Metabolites and Pathways Strategy initiative phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylinositol phosphatidylserine phosphatidylthreonine RAW 264.7 macrophage cell line murine resident peritoneal macrophages very long-chain fatty acid Ether-linked phospholipids may contain either an alkyl ether or vinyl ether bond at the sn-1 position of the glycerol backbone. These lipids are mostly found in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a variety of mammalian cell types, including macrophages (1Sugiura T. Nakajima M. Sekiguchi N. Nakagawa Y. Waku K. Different fatty chain compositions of alkenylacyl, alkylacyl and diacyl phospholipids in rabbit alveolar macrophages: high amounts of arachidonic acid in ether phospholipids.Lipids. 1983; 18: 125-129Crossref Scopus (96) Google Scholar, 2Akoh C.C. Chapkin R.S. Composition of mouse peritoneal macrophage phospholipid molecular species.Lipids. 1990; 25: 613-617Crossref PubMed Scopus (29) Google Scholar, 3Gaposchkin D.P. Zoeller R.A. Plasmalogen status influences docosahexaenoic acid levels in a macrophage cell line: insights using ether lipid-deficient variants.J. Lipid Res. 1999; 40: 495-503Abstract Full Text Full Text PDF PubMed Google Scholar). It is widely accepted that in most tissues, ether-linked PC exists mostly as plasmanylcholine (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine), with the exception of myocardia, whereas PE exists as plasmenylethanolamine. (1-O-alk-1’-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Vinyl-ether bearing phospholipids are also known as plasmalogens. In plasmalogens, the sn-2 position is usually occupied by PUFAs. Typically, the sn-1 positions in plasmanyl and plasmenyl lipids are occupied by either 16 or 18 carbon ether or vinyl ether moieties. Plasmalogen phospholipids affect membrane fluidity and fusion and the ether lipids alterations are associated with several cellular dysfunctions and diseases such as Alzheimer's, Down syndrome, and cerebro-hepato-renal (Zellweger) syndrome (4Brites P. Waterham H.R. Wanders R.J.A. Functions of plasmalogens in health and disease.Biochim. Biophys. Acta. 2004; 1636: 219-231Crossref PubMed Scopus (305) Google Scholar, 5Gorgas K. Teigler A. Komljenovic D. Just W.W. The ether lipid-deficient mouse: tracking down plasmalogen functions.Biochim. Biophys. Acta. 2006; 1763: 1511-1526Crossref PubMed Scopus (185) Google Scholar, 6Paltauf F. Ether lipids in biomembranes.Chem. Phys. Lipids. 1994; 74: 101-139Crossref PubMed Scopus (174) Google Scholar). Responsive cell types such as macrophages have relatively high content of plasmalogen lipids. Due to the high levels of PUFA in plasmalogens they are considered a storage for long-chain PUFAs and especially arachidonic acid, which can be released by plasmalogen-specific PLA2(iPLA2) into free arachidonic and docosahexaenoic acids which are further metabolized to second messenger molecules like eicosanoids and prostaglandins (7Farooqui A.A. Yang H.C. Horrocks L.A. Plasmalogens, phospholipase A2 and signal transduction.Brain Res. Brain Res. Rev. 1995; 21: 152-161Crossref PubMed Scopus (88) Google Scholar). Mass spectrometry has been the analytical method of choice for the characterization of lipid molecules, especially after introduction of the “soft” ionization techniques of MALDI (8Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4840) Google Scholar) and ESI (9Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6343) Google Scholar). The great potential of ESI-MS for analysis and characterization of nonvolatile and labile lipid molecules from biological extracts has been utilized extensively (9Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6343) Google Scholar, 10Pulfer M. Murphy R.C. Electrospray mass spectrometry of phospholipids.Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (733) Google Scholar, 11Han X. Yang J. Cheng H. Ye H. Gross R.W. Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Crossref PubMed Scopus (179) Google Scholar, 12Rouzer C.A. Ivanova P.T. Byrne M.O. Milne S.B. Marnett L.J. Brown H.A. Lipid profiling reveals glycerophospholipid remodeling in zymosan-stimulated macrophages.Biochemistry. 2007; 46: 6026-6042Crossref PubMed Scopus (43) Google Scholar), including the detection of some rare and unusual lipids (13Guan Z. Shengrong L. Smith D.C. Shaw W.A. Raetz C.H.R. Identification of N-acylphosphatidylserine molecules in eukaryotic cells.Biochemistry. 2007; 46: 14500-14513Crossref PubMed Scopus (39) Google Scholar). By employing phospholipid class separation and tandem mass spectrometry (LC/MS/MS), we were able to identify a number of previously not reported ether-linked phosphatidylserine (PS) and phosphatidic acid (PA) together with detection of ether phosphatidylinositol (PI) (14Lee T-C. Malone B. Buell A.B. Blank M.L. Occurrence of ether-containing inositol phospholipids in bovine erythrocytes.BBRC. 1991; 175: 673-678PubMed Google Scholar) and phosphatidylthreonine (PT) (15Mark-Malchoff D. Marinetti G.V. Hare G.D. Meisler A. Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts.Biochemistry. 1978; 17: 2684-2688Crossref PubMed Scopus (13) Google Scholar) in extracts from RAW264.7 cells, foam cells, murine bone marrow derived macrophages (BMDM) and murine resident peritoneal macrophages (RPM). In the course of the analysis, we also identified some plasmanyl and plasmenyl PC and PE lipids containing very long-chain fatty acids (VLCFA) (24 C-atom and more). The motivation of this study was to identify novel and atypical lipid species in a variety of commonly used macrophage preparations. This study is not intended to provide a comprehensive accounting of all of the glycerophospholipid species in these cells, which continues as an ongoing project. 37:4 PI (1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1’-myo-inositol)), 38:4e PI (1-octadecyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho- (1’-myo-inositol)), 34:1e PC (1-hexadecyl-2-(9Z-octadecenoyl)- sn-glycero-3-phosphocholine), 34:1p PC (1-(1Z-octadecenyl)- 2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine), 34:1e PE (1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine), and 34:1p PE (1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). HPLC grade solvents were purchased from VWR (West Chester, PA) and used without further purification. BMDM, foam, and RAW 264.7 cells were acquired as cell pellets prepared by established protocols. Briefly, BMDMs were harvested from tibias and femurs of 2-month-old C57BL6 male mice. They were suspended in Bone Marrow-Derived Macrophage Growth Medium (BMDMGM), plated on 100 mm Petri dishes and maintained at 37°C in a humidified incubator for 4 days. On day 4, cells were washed with RPMI medium and maintained at 37°C in BMDMGM for 2 more days, after which the macrophages were plated on 100 mm tissue culture dishes at a density of 5 × 106 per plate. Foam cells were elicited from male mice (B6.129S7-LDLrtm1 Her/J) (3–5 weeks of age; Jackson Laboratory) after being placed on a high-cholesterol diet (# TD96121, Harlan Teklad) for 10 weeks. Mice were injected with 2.5 ml thioglycollate intraperitoneally and foam cells were harvested 4 days post injection. RAW 264.7 cells were maintained essentially as described elsewhere (12Rouzer C.A. Ivanova P.T. Byrne M.O. Milne S.B. Marnett L.J. Brown H.A. Lipid profiling reveals glycerophospholipid remodeling in zymosan-stimulated macrophages.Biochemistry. 2007; 46: 6026-6042Crossref PubMed Scopus (43) Google Scholar). All studies involving animals were conducted with the approval of the Institutional Animal Care and Use Committee of Vanderbilt University. Female ICR (CD-1) mice (25–30g) were obtained form Harlan (Indianapolis, IN). Cells were obtained by peritoneal lavage as described previously (16Rouzer C.A. Marnett L.J. Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus.J. Biol. Chem. 2005; 280: 26690-26700Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and suspended at a density of 2–3 × 106 cells/ml (cells from one mouse per 2 ml) in Minimal Essential Alpha Medium supplemented with GlutaMax (Gibco), 10% heat-inactivated fetal calf serum (Atlas Biologicals, Norcross, GA), and 100 units/ml penicillin and 0.10 mg/ml streptomycin (Sigma, St.Louis, MO) (α-MEM/FCS). The cell suspension was plated on 60 mm tissue culture plates at a density of 6ml/plate and incubated for 2 h at 37°C in a humidified 5% CO2 atmosphere. Nonadherent cells were removed by washing the plates four times with PBS, and the cultures were then incubated overnight in fresh α-MEM/FCS. Phospholipids were extracted using a modified Bligh and Dyer procedure (17Milne S. Ivanova P. Forrester J. Brown H.A. Lipidomics: an analysis of cellular lipids by ESI-MS.Methods. 2006; 39: 92-103Crossref PubMed Scopus (152) Google Scholar). Typically, between 1 and 3 × 106 cells per sample were used in this protocol. The method is suitable for extraction from cell culture plates after aspirating the medium and washing the adhered cells twice with 5 ml ice cold 1× PBS. Cells are then scraped in 1 ml of 1× PBS, and centrifuged (600 g, 4°C, 5 min). PBS is aspirated and the cell pellet is extracted with 800 μl of cold 0.1 N HCl: MeOH (1:1) and 400 μl of cold CHCl3 with vortexing (1 min) followed by centrifugation (5 min, 4°C, 18,000 g). The lower organic phase is then isolated and solvent evaporated (Labconco Centrivap Concentrator, Kansas City, MO). The extraction procedure includes acidification (0.05N HCl) in the aqueous phase that aids in the lysolipids and acidic lipids recovery. Class separation of glycerophospholipids was achieved by the use of a previously published LC/MS technique (18Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (137) Google Scholar). After extraction and solvent evaporation (as described above) the resulting lipid film is dissolved in 100 μl of IPA:Hexane:100 mM NH4CO2H(aq) 58:40:2 (mobile phase A). For the lipid screens, we utilized an Applied Biosystems/ MDS SCIEX 4000 Q TRAP hybrid triple quadrupole/ linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA). Coupled to this instrument were a Shimadzu (Shimadzu Scientific Instruments, Inc., Columbia, MD) HPLC system consisting of a SCL 10 AVP controller, two LC 10 ADVP pumps and a CTC HTC PAL autosampler (Leap Technologies, Carrboro, NC). All samples shown were separated on a Phenomenex (Phenomenex, Torrance, CA) Luna Silica column (2 × 250 mm, 5 micron particle size) using a 20 μl sample injection. Lipids were separated using a binary gradient program consisting of IPA:Hexane:100 mM NH4CO2H(aq) 58:40:2 (mobile phase A), and IPA:Hexane:100 mM NH4CO2H(aq) 50:40:10 (mobile phase B). The following LC gradient was used: 0–5min, B = 50%; 5–30min, B = 50%–100%; 30–40min, B = 100%; 40–41min, B = 100%–50%; 41–50min, B = 50%. The mobile phase was infused at a flow rate of 0.3 ml/min. The MS spectra were acquired in negative ionization mode using a turbo spray source operated at 450°C with an ion voltage of –3500V, and nitrogen as curtain and nebulizer gas. The curtain gas was 30 L/h, and ion source gas 1 and 2 were both 50 L/h. The declustering potential was −110 V and the collision energy was −5 V. Scan type: EMS, unit resolution for Q1; Scan rate: 1000 amu/s; Scan range from m/z 350–1200, with the ion trap set for dynamic fill time. Large scale screening and identification of glycerophospholipids was accomplished by LC/MS/MS analysis using the information dependent analysis (IDA) software option within the Analyst software package (Applied Biosystems) and individual fragmentation. In conjunction with the above mentioned LC/MS phospholipid separation protocol, MS/MS experiments were run over the following m/z regions: 350-600 m/z, 600-800 m/z, 800-1000 m/z, and 1000-1200 m/z. Using this technique, upwards of 500 MS/MS data files can be collected per injection (or 2000 per sample if all four spectra regions are analyzed). Identification of lipid species is then made by analysis of retention time and comparison to previously published fragmentation patterns (19Hsu F-F. Turk J. Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal.J. Am. Soc. Mass Spectrom. 2000; 11: 797-803Crossref PubMed Scopus (99) Google Scholar, 20Hsu F-F. Turk J. Charge-remote and charge-driven fragmentation processes in diacyl glycerophosphoethanolamine upon low-energy collisional activation: a mechanistic proposal.J. Am. Soc. Mass Spectrom. 2000; 11: 892-899Crossref PubMed Scopus (162) Google Scholar, 21Hsu F-F. Turk J. Differentiation of 1-O-alk-1’-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids by multiple-stage linear ion-trap mass spectrometry with electrospray.J. Am. Soc. Mass Spectrom. 2007; 18: 2065-2073Crossref PubMed Scopus (62) Google Scholar, 22Hsu F-F. Turk J. Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study.J. Am. Soc. Mass Spectrom. 2000; 11: 986-999Crossref PubMed Scopus (225) Google Scholar) and chemically defined standards obtained from Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL). Direct infusion mass spectral analysis was performed on an Applied Biosystems/ MDS SCIEX 4000 Q TRAP hybrid triple quadrupole linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA). The instrument was equipped with a Harvard Apparatus syringe pump and an electrospray ion source. Samples were analyzed at an infusion rate of 10 μl/min in negative ionization mode over the range of m/z 350 to 1200. Data were collected with the Analyst software package (Applied Biosystems). Collision induced fragmentation of targeted lipid species was accomplished using a collision energy appropriate for the specific lipid class (−35 to −45 V). Glycerophospholipids are separated using normal-phase chromatography by class according to their polarity. Ether-linked phospholipids elute faster than their diacyl counterparts and thus are separated on the Luna silica column. Under the described chromatographic conditions, the phospholipids elute in the following order: PG< PI, PE< PA< PS/PT< PC. Figure 1A, B, and C shows the extracted ion chromatograms for PG and PI, showing the faster elution times of the ether-linked PI (Fig. 1A) compared with diacyl-PI (Fig. 1C). Odd carbon fatty acid containing 37:4 PI elutes after 38:4 PI, thus being distinguished from ether PI with the same mass (37:4 PI would have the same mass as 38:4e PI). Because the presence of ether-PI in macrophages has not been previously reported, the identification was confirmed by comparison of retention time and fragmentation spectra to a chemically defined synthetic standard (38:4e PI). Retention time confirmation was achieved by addition of synthetic 38:4e PI standard to an RPM cell extract and analyzed by LC/MS. Figure 1 shows the extracted ion chromatograms of the [M-H]− ion at m/z 871.6 characteristic for the 38:4ePI from the RPM extract (Fig. 1A) compared with the one from an RPM extract with the addition of the 38:4e PI standard (Fig. 1B). Structural identification was accomplished by LC/MS/MS. The synthetic standard with a formula of C47H85O12P has a molecular ion [M-H]− at m/z 871.57 and its fragmentation spectrum is shown on Fig. 2. The major identified peaks correspond to the 38:4e PA, 18:0e lysoPA and its dehydrated form at m/z 709.5, 423.3, and 405.3, respectively, due to loss of the headgroup. Another key fragment belongs to 18:0e lysoPI at m/z 585.7 and its dehydrated form at m/z 567.6 from losses of 20:4 fatty acid at sn-2 as a ketene and as an acid, respectively. These ions reflect the 20:4 substituent at sn-2, including the carboxylate ion detected at m/z 303.1. Fragments corresponding to the headgroup are also present at m/z 259.3 and 241. As shown in Figs. 3 and 4, fragmentation spectra from naturally occurring ether-PI are detected in RPM and foam cell extracts. The MS/MS spectrum of the molecular ion [M-H]− at m/z 871.6 from RPM cell extracts (Fig. 3) has a very similar pattern, but contains more fragment peaks, clearly revealing the presence of more than one pair of fatty acid combinations for the 38:4e PI. According to the fragmentation pattern, this peak (at m/z 871.6) is composed of 18:0e/20:4; 18:1e/20:3; and 16:0e/22:4 PI. Similarly, ether PI species of different chemical composition were detected in the other investigated cell types. An example is shown in Fig. 4, depicting the MS/MS data from a naturally occurring 40:4e PI in foam cells.Fig. 2MS/MS spectra of a 38:4e PI (18:0e/20:4) synthetic standard. Fragmentation of the chemically defined PI standard yielded a plethora of lyso PA, lyso PI, and fatty acid fragments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3MS/MS spectra of naturally occurring 38:4e PI lipid from RPM cell extract. Fragmentation of the 871 m/z peak eluting prior to 38:4 PI yielded fragments consistent with the pattern observed in the synthetic standard. Additional fragments were also identified that were assigned to fatty acid combinations other than 18:0e/20:4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4MS/MS spectra of a naturally occurring foam cell 40:4e PI lipid. MS/MS analysis of the 899 m/z peak eluting prior to the 40:X series of diacyl PI lipids was shown to be primarily composed of a 18:0e and 22:4 ether/fatty acid combination.View Large Image Figure ViewerDownload Hi-res image Download (PPT) During the comprehensive lipid profiling of these cells, another two classes of ether-containing glycerophospholipids were identified, which have not been reported previously in macrophages, but have been identified in other biological tissues (23Deeley J.M. Thomas M.C. Truscott R.J.W. Mitchell T.M. Blanksby S.J. Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques.Anal. Chem. 2009; 81: 1920-1930Crossref PubMed Scopus (47) Google Scholar, 24Kaneshiro E.S. Guo Z. Sul D. Kallam K.A. Jayasimhulu K. Beach D.H. Characterization of Pneumocystis carinii and rat lung lipids: glyceryl ethers and fatty alcohols.J. Lipid Res. 1998; 39: 1907-1917Abstract Full Text Full Text PDF PubMed Google Scholar). The presence of ether-PA was established in the chromatographic peak fraction from RAW 264.7 and foam cells. A representative MS/MS spectrum of a naturally occurring 32:0e PA is shown in Fig. 5. The spectrum contains a pair of prominent ions at m/z 395.1 and 377.1 arising from the loss of 16:0 fatty acid as a ketene and acid, respectively. Another pair of ions at m/z 423.2 and 405.3 corresponds to the loss of 14:0 fatty acid as a ketene and acid. These ions reflect the 14:0 and 16:0 fatty acid substituents at sn-2, and the ions reflecting the radyl group at sn-1 are not available. The presence of carboxylate ions at m/z 255.2 and 227.2 corresponding to 16:0 and 14:0 fatty acids, respectively, confirm the presence of 18:0e/14:0 and 16:0e/16:0 PA in the molecular ion at m/z 633.6. Proposed ether-linked PA species detected in RAW 264.7 and one in foam cell extracts are presented in Table 1.TABLE 1Proposed ether- and vinyl ether-linked PA and PS molecular species detected in RAW, BMDM, and foam cellsm/zRAWBMDMFoamPA63316:0e/16:018:0e/14:065916:0e/18:118:0e/16:166116:0e/18:018:0e/16:068118:0e/20:468718:0e/18:170716:0e/22:538:4p/38:5e73720:0e/20:4PS79616:0e/22:418:0e/20:418:0e/20:479816:0e/20:318:0e/20:382418:0e/22:418:0e/22:418:0e/22:420:0e/20:4More species are identified in RAW 264.7 cell extracts due to the extensive work on these cells compared with others. Open table in a new tab More species are identified in RAW 264.7 cell extracts due to the extensive work on these cells compared with others. Similarly, a number of ether-PS species were identified in other macrophage extracts (Table 1). The representative MS/MS spectrum on Fig. 6 shows fragments consistent with 18:0e/22:4 PS. Phosphatidylserine shows a strong molecular ion [M-H]− at m/z 824.6, as well as a prominent [M-H-87]− at m/z 737.4, indicating the loss of the serine headgroup and identical to 40:4e PA. The ion at m/z 423.3 reflects the loss of 22:4 fatty acid at sn-2 position as a ketene [M-H-87-R2CH = C = O]−, whereas the ion at m/z 405.2 indicates the loss of 22:4 fatty acid as an acid [M-H-87-R2COOH]−. 22:4 Carboxylate ion at m/z 331.1 is also present. Here again, the ions corresponding to the radyl substituents at sn-1 are not detected in agreement with previously shown fragmentation of plasmanyl phospholipids (21Hsu F-F. Turk J. Differentiation of 1-O-alk-1’-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids by multiple-stage linear ion-trap mass spectrometry with electrospray.J. Am. Soc. Mass Spectrom. 2007; 18: 2065-2073Crossref PubMed Scopus (62) Google Scholar). We hypothesize that the ether and vinyl ether PA, PI, and PS glycerophospholipids reported above are direct or indirect products from the biosynthesis of plasmanyl and plasmenyl PC and PE glycerophospholipids. This reaction pathway is a combination of the traditional biosynthesis routes for plasmanyl and plasmenyl lipids (25Lee T.C. Biosynthesis and possible biological functions of plasmalogens.Biochim. Biophys. Acta. 1998; 1394: 129-145Crossref PubMed Scopus (95) Google Scholar) and the Kennedy pathway (26Kennedy E.P. Sailing to Byzantium.Annu. Rev. Biochem. 1992; 61: 1-28Crossref PubMed Scopus (23) Google Scholar). PT is a phospholipid that has been identified and reported as present in significant amounts in some cultured cells (15Mark-Malchoff D. Marinetti G.V. Hare G.D. Meisler A. Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts.Biochemistry. 1978; 17: 2684-2688Crossref PubMed Scopus (13) Google Scholar, 27Heikinheimo L. Somerharju P. Translocation of phosphatidylthreonine and–serine to mitochondria diminishes exponentially with increasing molecular hydrophobicity.Traffic. 2002; 3: 367-377Crossref PubMed Scopus (25) Google Scholar, 28Mitoma J. Kasama T. Furuya S. Hirabayashi Y. Occurrence of an unusual phospholipid, phosphatidyl-L-threonine, in cultured hippocampal neurons.J. Biol. Chem. 1998; 273: 19363-19366Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Reports of these lipids have not been published for macrophage cell extracts. Structural analogy with PS makes its detection impossible without careful class separation via LC/MS/MS. The method we employ for phospholipid LC analysis allows its separation from PS and other glycerophospholipids. Using the gradient and the silica column described above, PT elutes after PS, although some ether-linked PS species coelute with PT species, but differs in its fragmentation spectrum used for the identification. As in the case of PS, PT ionizes well and is analyzed in negative ionization mode. The fragmentation spectrum shown in Fig. 7 reveals the major peaks identified in the molecular ion peak at m/z 850.5 (from BMDM). The most prominent ion present in the spectrum is the one from a neutral loss of the headgroup ([M-H-101]−) yielding the corresponding phosphatidic acid at m/z 749.4, which is consistent with [M-H]− ion of 18:0/22:5 PA. The ions at m/z 483.4 ([M-H-101-R1CH = C = O]−) and 437.3 ([M-H-101-R2CH = C = 0]−) are a result of losing fatty acid substituents at sn-1 and sn-2 as ketenes, whereas the ions at m/z 465.3 ([M-H-101-R1COOH]−) and 419.3 ([M-H-101-R2COOH]−) illustrate the loss of the corresponding substituents as acids. The carboxylic ions at m/z 283.1 and 329.4 are characteristic for the two fatty acid substituents at sn-1 and sn-2. As mentioned before, the molecular ion peak at m/z 850.5 also contains some PS (ether-linked) and the peak assignable to a PA fragment (m/z 763.52) coming from PS is noted by an asterisk in Fig. 7. All of the above peak assignments are in good correlation with previously reported data for PS and PT mass spectral analyses (27Heikinheimo L. Somerharju P. Translocation of phosphatidylthreonine and–serine to mitochondria diminishes exponentially with increasing molecular hydrophobicity.Traffic. 2002; 3: 367-377Crossref PubMed Scopus (25) Google Scholar, 28Mitoma J. Kasama T. Furuya S. Hirabayashi Y. Occurrence of an unusual phospholipid, phosphatidyl-L-threonine, in cultured hippocampal neurons.J. Biol. Chem. 1998; 273: 19363-19366Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 29Hsu F-F. Turk J. Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: structural characterization and the fragmentation processes.J. Am. Soc. Mass Spectrom. 2005; 16: 1510-1522Crossref PubMed Scopus (111) Google Scholar). Over 30 PT species were identified in different macrophages (proposed phospholipid species presented in Table 2). Differences in the number of identified species are mainly due to the large number of RAW 264.7 cell extracts analyzed during the course of the work compared with the other types of macrophages.TABLE 2Proposed PT molecular species detected in RAW, BMDM, foam, and RPM cellsm/zRAWBMDMFoamRPM76016:1/17:016:1/17:016:0/17:116:0/17:116:0/17:178817:0/18:117:0/18:117:1/18:017:1/18:080018:0/18:218:1/18:180218:0/18:118:0/18:118:0/18:117:0/19:180418:0/18:018:0/18:081217:0/20:317:0/20:317:1/20:217:1/20:282418:1/20:318:0/20:418:0/20:482618:0/20:383417:0/22:617:0/22:617:0/22:683617:0/22:517:0/22:517:0/22:583817:0/22:417:0/22:484818:0/22:685018:0/22:5 Open table in a new tab A significantly large group of PC and PE species containing VLCFA with different degrees of unsaturation were identified in all of the analyzed macrophages. The presence of these fatty acids is usually associated with peroxisomal β-oxidation defects and neurodegenerative disorders (30Kemp S. Valianpour F. Denis S. Ofman R. Sanders R-J. Mooyer P. Barth P.G. Wanders R.J.A. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodistrophy.Mol. Genet. Metab. 2005; 84: 144-151Crossref PubMed Scopus (72) Google Scholar). These species have never been detected in macrophages before. Multiple species of PC from RAW 264.7 cell extracts contain 24:X and 26:X fatty acids in diacyl, plasmanyl, and plasmenyl phosphatidylcholines, whereas VLCFA PC from the other types of macrophages mostly consist of 24:X fatty acids. 24:X and 26:X fatty acids are present in diacyl, plasmanyl, and plasmenyl PE from all types macrophages (Tables 3 and 4). Here again, the majority of identified species are within RAW264.7 cell extracts as a result of the most number of samples analyzed. During the process of identification by tandem mass spectrometry, multiple ions corresponding to the fatty acid substituents and the lyso components of the molecules were observed all in agreement with previously published data (21Hsu F-F. Turk J. Differentiation of 1-O-alk-1’-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids by multiple-stage linear ion-trap mass spectrometry with electrospray.J. Am. Soc. Mass Spectrom. 2007; 18: 2065-2073Crossref PubMed Scopus (62) Google Scholar, 31Kayganich K.A. Murphy R.C. Fast atom bombardment tandem mass spectrometric identification of diacyl, alkylacyl, and alk-1-enylacyl molecular species of glycerophosphoethanolamine in human polymorphonuclear leukocytes.Anal. Chem. 1992; 64: 2965-2971Crossref PubMed Scopus (99) Google Scholar, 32Yang K. Zhao Z. Gross R.W. Han X. Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species.PLoS One. 2007; 2: e1368Crossref PubMed Scopus (43) Google Scholar). As with all plasmanyl PE and PC, there were no fragments corresponding to the alkyl moiety, but just fragments arising from the loss of the acyl moiety, thus creating ether-containing lyso lipid fragments and their dehydrated forms. Both PE and PC were analyzed in negative ionization mode. PCs were identified as formate adducts. Analysis in negative mode ionization affords information on the fatty acid composition as well as the headgroup. Not only were VLCFA-containing PC and PE identified, but also the presence of unusual ether moieties was detected. Usually, the sn-1 positions in plasmanyl and plasmenyl lipids are occupied by either 16 or 18 carbon ether or vinyl ether moieties, whereas in these cell extracts, we detected species containing 14, 16, 18, 20, 22, and 24 carbon ether or vinyl ether substituents. Mass spectral analysis confirmed their structure following analysis of fragmentation ions corresponding to the acyl substituent as a carboxylate ion and only the loss of the acyl group as acid or ketene (and therefore forming a ether-lysoPC and ether-lysoPE).TABLE 3.Proposed very long-chain diacyl, plasmanyl, and plasmenyl PC molecular speciesaPC lipids were identified as formate adducts.m/zRAWBMDMFoamRPM86416:0e/24:616:0e/24:686616:0e/24:516:0e/24:516:0e/24:520:0e/20:520:0e/20:586816:0e/24:416:0e/24:416:0e/24:416:0e/24:420:0e/20:420:0e/20:420:0e/20:420:0e/20:487016:0e/24:320:0e/20:387216:0e/24:216:0e/24:220:0e/20:287416:0e/24:116:0e/24:120:0e/20:120:0e/20:122:0e/18:187616:1/24:687816:0/24:616:0/24:616:0/24:616:1/24:588016:0/24:516:0/24:516:0/24:516:0/24:516:1/24:416:1/24:416:1/24:488216:0/24:416:0/24:416:0/24:416:0/24:416:1/24:389218:0e/24:620:0e/22:689418:0e/24:518:0e/24:518:0p/24:420:0e/22:520:0e/22:520:0e/22:522:0p/20:489616:0e/26:418:0e/24:418:0e/24:418:0e/24:420:0e/22:420:0e/22:420:0e/22:422:0e/20:422:0e/20:489818:0e/24:320:0e/22:322:0e/20:390016:0e/26:218:0e/24:290218:0e/24:192022:0e/22:692218:0e/26:520:0e/24:522:0e/22:522:0e/22:592418:0e/26:420:0e/24:420:0e/24:422:0e/22:424:0e/20:492620:0e/24:322:0e/22:324:0e/20:394822:0e/24:624:0e/22:695022:0e/24:596622:4/24:4a PC lipids were identified as formate adducts. Open table in a new tab TABLE 4Proposed very long chain diacyl, plasmanyl, and plasmenyl PE molecular speciesm/hRAWBMDMFoamRPMm/zRAWBMDMFoamRPM77816:0e/24:580420:0e/22:620:0e/22:620:0e/22:618:0e/22:516:0p/24:416:0p/26:516:0p/26:516:0p/26:516:0p/24:418:0p/22:416:0p/24:418:0p/24:518:0p/24:518:0p/24:518:0p/22:420:0p/20:420:0p/22:520:0p/22:520:0p/22:520:0p/22:520:0p/20:480618:0e/24:518:0e/24:518:0e/24:578016:0e/24:420:0e/22:520:0e/22:520:0e/22:520:0e/22:518:0e/22:416:0p/26:420:0e/20:420:0e/20:418:0p/24:418:0p/24:416:0p/24:320:0p/22:420:0p/22:420:0p/22:420:0p/22:418:0p/22:322:0p/20:422:0p/20:422:0p/20:478216:0e/24:381017:2/24:118:0e/22:381616:1/26:620:0e/20:318:2/24:516:0p/24:218:1/24:618:0p/22:281818:2/24:418:2/24:418:2/24:478416:0e/24:216:0e/24:218:1/24:518:1/24:518:1/24:518:0e/22:218:0e/22:218:0/24:618:0/24:618:0/24:616:0p/24:116:0p/24:116:1/26:518:0p/22:118:0p/22:182016:1/26:418:1/24:420:0p/20:120:0p/20:116:0/26:518:0/24:578614:0e/26:118:2/24:316:0e/24:118:1/24:418:1/24:479016:1/24:518:0/24:518:0/24:516:0/24:682216:1/26:379216:1/24:416:0/26:416:0/24:516:0/24:518:1/24:318:1/24:379416:1/24:318:0/24:418:0/24:418:0/24:416:0/24:416:0/24:416:0/24:482416:1/26:279616:1/24:218:2/24:116:0/24:318:1/24:280016:1/24:118:0/24:316:0/24:082816:1/26:080216:0p/26:618:1/24:018:0p/24:618:0p/24:618:0/24:120:0p/22:685018:1/26:417:1/24:618:1/26:320:3/24:120:4/24:0 Open table in a new tab Despite the widely held notion that odd carbon fatty acids only exist in plants and some lower organisms, we detected multiple species in almost all of the analyzed glycerophospholipid classes in all types of macrophages (also in multiple human tissue, astrocytoma, and other cancer cell extracts, data not shown). Their molecular weight is equal to the alkyl ether species of the same phospholipid class, but can be distinguished from them by their elution properties and the fragmentation spectra. Unlike ether glycerophospholipids that do not produce carboxylate anions for the sn-1 (ether-bound) moiety, odd-carbon containing phospholipids show prominent carboxylate anions for the corresponding fatty acids in addition to the odd carbon lyso glycerophospholipid fragments. In most cell types analyzed, lipids containing at least one odd carbon fatty acid account for approximately 15% (by number) of the total number of phospholipids identified by MS/MS fragmentation. The evidence presented here for the existence of these unusual glycerophospholipid classes in macrophages will initiate new endeavors to determine the biological functions of these species. The presence of a relatively large number of ether-containing phospholipid species (from most classes) could contribute to the ability of these cells to produce potent biological mediators like platelet-activating factor, free PUFAs, or very long-chain fatty acids upon stimulation. In addition, the increased presence of plasmalogen phospholipids in tissues has been correlated with malignancy and metastatic properties of human cancers (33Smith R.E. Lespi P. Di Luca M. Bustos C. Marra F.A. de Alaniz M.J.T. Marra C.A. A reliable biomarker derived from plasmalogens to evaluate malignancy and metastatic capacity of human cancers.Lipids. 2008; 43: 79-89Crossref PubMed Scopus (44) Google Scholar). The authors thank Dr. Carol Rouzer for providing RPM cells and Andrew Goodman for excellent technical assistance. We thank members of the LIPID MAPS consortium for providing some of the foam and BMDM cells used for the characterization of lipid species. This work was supported in part by a large scale consortium grant from the National Institutes of Health U54 GM069338.
Glycerophospholipids
Glycerophospholipids
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Summary Microorganisms, such as Pseudomonas putida , utilize specific physical properties of cellular membrane constituents, mainly glycerophospholipids, to (re‐)adjust the membrane barrier to environmental stresses. Building a basis for membrane composition/function studies, we inventoried the glycerophospholipids of different Pseudomonas and challenged membranes of growing cells with n‐butanol. Using a new high‐resolution liquid chromatography/mass spectrometry (LC/MS) method, 127 glycerophospholipid species [e.g. phosphatidylethanolamine PE(32:1)] with up to five fatty acid combinations were detected. The glycerophospholipid inventory consists of 305 distinct glycerophospholipids [e.g. PE(16:0/16:1)], thereof 14 lyso ‐glycerophospholipids, revealing conserved compositions within the four investigated pseudomonads P. putida KT2440, DOT‐T1E, S12 and Pseudomonas sp. strain VLB120. Furthermore, we addressed the influence of environmental conditions on the glycerophospholipid composition of Pseudomonas via long‐time exposure to the sublethal n‐butanol concentration of 1% (v/v), focusing on: (i) relative amounts of glycerophospholipid species, (ii) glycerophospholipid head group composition, (iii) fatty acid chain length, (iv) degree of saturation and (v) cis/trans isomerization of unsaturated fatty acids. Observed alterations consist of changing head group compositions and for the solvent‐sensitive strain KT2440 diminished fatty acid saturation degrees. Minor changes in the glycerophospholipid composition of the solvent‐tolerant strains P. putida S12 and Pseudomonas sp. VLB120 suggest different strategies of the investigated Pseudomonas to maintain the barrier function of cellular membranes.
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Glycerophospholipids
Pseudomonas putida
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Human milk provides a complex mixture of animal lipids, whereas the fat supply of most modern infant formula is based on vegetable oils. We studied the effects of breast-feeding and of feeding infant formula either without or with dairy goat lipids on the composition of infant plasma glycerophospholipids.Healthy-term infants were randomized double blind to feeding with infant formula based on whole goats' milk (GIF, approximately 60% milk fat and 40% vegetable oils) or a control cows' milk infant formula based on vegetable oils (VIF) from 2 weeks after birth. A reference group of fully breast-fed infants was also followed. At the age 4 months, blood samples were collected and plasma glycerophospholipids were analyzed with liquid chromatography coupled to triple quadrupole mass spectrometry.The group of breast-fed infants showed significantly higher contents of glycerophospholipid species containing sn-2 palmitic acid [PC(16:0/16:0) and PC(18:0/16:0)] and significantly higher contents of glycerophospholipid species containing long-chain polyunsaturated fatty acids than infants in both formula groups. The GIF group demonstrated significantly higher glycerophospholipid species containing myristic acid [LPC(14:0), PC(14:0/18:1), PC(16:0/14:0)] and palmitoleic acid [LPC(16:1), PC(16:0/16:1), and PC(16:1/18:1)] than the VIF group.We conclude that breast-feeding induces marked differences in infant plasma glycerophospholipid profiles compared with formula feeding, whereas the studied different sources of formula fat resulted in limited effects on plasma glycerophospholipids.
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A detailed analysis of the (35)Cl/(37)Cl isotope shifts induced in the 128.8 MHz (195)Pt NMR resonances of [PtCl(n)(H(2)O)(6 - n)](4 - n) complexes (n = 6,5,4) in acidic solution at 293 K, shows that the unique isotopologue and isotopomer distribution displayed by the resolved (195)Pt resonances, serve as a fingerprint for the unambiguous identification and assignment of the isotopic stereoisomers of [PtCl(5)(H(2)O)](-) and cis/trans-[PtCl(4)(H(2)O)(2)].
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Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols with this approach.
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We have performed a spectral line survey called EMoCA toward Sagittarius B2(N) between 84 and 114 GHz with ALMA. Line intensities of the main isotopic species of ethyl cyanide and its singly 13C-substituted isotopomers observed toward the hot molecular core Sgr B2(N2) suggest that the doubly 13C-substituted isotopomers should be detectable also. We want to determine the spectroscopic parameters of all three doubly 13C-substituted isotopologues of ethyl cyanide to search for them in our ALMA data. We investigated the laboratory rotational spectra of the three species between 150 and 990 GHz. We searched for emission lines produced by these species in the ALMA spectrum of Sgr B2(N2). We modeled their emission as well as the emission of the 12C and singly 13C-substituted isotopologues assuming local thermodynamic equilibrium. We identified more than 5000 rotational transitions, pertaining to more than 3500 different transition frequencies, in the laboratory for each of the three isotopomers. The quantum numbers reach J ~ 115 and K_a ~ 35, resulting in accurate spectroscopic parameters and accurate rest frequency calculations beyond 1000 GHz for strong to moderately weak transitions of either isotopomer. All three species are unambiguously detected in our ALMA data. The 12C/13C column density ratio of the isotopomers with one 13C atom to the ones with two 13C atoms is about 25. Ethyl cyanide is the second molecule after methyl cyanide for which isotopologues containing two 13C atoms have been securely detected in the interstellar medium. The model of our ethyl cyanide data suggests that we should be able to detect vibrational satellites of the main species up to at least v_19 = 1 at 1130 K and up to v_13 + v_21 = 2 at 600 K for the isotopologues with one 13C atom in our present ALMA data. Such satellites may be too weak to be identified unambiguously for isotopologues with two 13C atoms.
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