Lecithin pp 3-16 | Cite as

Modern Techniques for the Fractionation and Purification of Phospholipids from Biological Materials

  • Lloyd A. Horrocks
  • Laura L. Dugan
  • Cheryl J. Flynn
  • Gianfrancesco Goracci
  • Serena Porcellati
  • Young Yeo
Part of the Advances in Behavioral Biology book series (ABBI, volume 33)


Glycerophospholipids have important roles in biological processes. In addition to forming the hydrophobic backbone of cell membranes, glycerophospholipids also participate actively in signal transduction. A number of receptors respond to agonist-binding by activation of a phospholipase C that hydrolyzes PtdIns 4,5-P2 to InsP3 and diacylglycerols (Berridge and Irvine, 1984; Berridge et al., 1985; Berridge, 1986; Nishizuka, 1984a, b; Hirasawa and Nishizuka, 1985; Abdel-Latif et al., 1985; Akhtar and Abdel-Latif, 1986). The InsP3 may increase cytosolic Ca2+ concentrations, thus activating a protein kinase and other reactions. The diacylglycerol stimulates protein kinase C.


Platelet Activate Factor Phospholipid Class Methyl Formate Rabbit Platelet High Performance Liquid Chromatography Separation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abdel-Latif, A. A., Smith, J. P., and Akhtar, R. A., 1985, Polyphosphoinositides and muscarinic cholinergic and alpha-adrenergic receptors in the iris smooth muscle., in: “Inositol and Phosphoinositides: Metabolism and regulation.,” Bleasdale, J. E., Eichberg, J., and Hauser, G., eds., Humana Press, Clifton, NJ.Google Scholar
  2. Akhtar, R. A. and Abdel-Latif, A. A., 1986, Surgical sympathetic denervation increases alpha 1-adrenoceptor-mediated accumulation of myo-inositol trisphosphate and muscle contraction in rabbit iris dilator smooth muscle., J. Neurochem., 46:96.PubMedCrossRefGoogle Scholar
  3. Albert, D. H. and Snyder, F., 1983, Biosynthesis of 1-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-acy1-sn-glycero-3-phosphocholine by rat alveolar macrophages. Phospholipase A2 and acetyltransferase activities during phagocytosis and ionophore stimulation, J. Biol. Chem., 258:97.PubMedGoogle Scholar
  4. Andreesen, R., Modolell, M., Oepke, G. H., Common, H., Lohr, G. W., and Munder, P. G., 1982, Studies on various parameters influencing leukemic cell destruction by alkyl-lysophospholipids, Anticancer Res., 2:95.PubMedGoogle Scholar
  5. Andreesen, R., Modolell, M., Weltzien, H. U., Eibl, H., Common, H. H., Lohr, G. W., and Munder, P. G., 1978, Selective destruction of human leukemic cells by alkyl-lysophospholipids, Cancer Res., 38:3894.PubMedGoogle Scholar
  6. Arthur, G., Mock, T., Zaborniak, C., and Choy, P. C., 1985, The distribution and acyl composition of plasmalogens in guinea pig heart, Lipids, 20:693.PubMedCrossRefGoogle Scholar
  7. Benveniste, J., 1985, PAF acether (Platelet activating factor), Adv. Prostaglandin Thromboxane Res., 13:11.Google Scholar
  8. Berdel, W. E., Bausert, W. R., Fink, U., Rastetter, J., and Munder, P. G., 1981, Anti-tumor action of alkyl-lysophospholipids, Anticancer Res., 1:345.PubMedGoogle Scholar
  9. Berridge, M. J., 1986, Intracellular signalling through inositol trisphosphate and diacylglycerol., Hoppe-Seyler’s Z. Physiol. Chem., 367:447.Google Scholar
  10. Berridge, M. J. and Irvine, R. F., 1984, Inositol trisphosphate, a novel second messenger in cellular signal transduction., Nature, 312:315.PubMedCrossRefGoogle Scholar
  11. Berridge, M. J., Brown, K. D., Irvine, R. F., and Heslop, J. P., 1985, phosphoinositides and cell proliferation., J. Cell Sci., 1985(S3):187.CrossRefGoogle Scholar
  12. Blank, M. L., Robinson, M., Fitzgerald, V., and Snyder, F., 1984, Novel quantitative method for determination of molecular species of phospholipids and diglycerides, J. Chromatogr., 298:473.PubMedCrossRefGoogle Scholar
  13. Bligh, E. G. and Dyer, W. J., 1959, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37:911.PubMedCrossRefGoogle Scholar
  14. Boulton, A. A., Baker, G. B., and Horrocks, L. A., 1987, “Neuromethods,” vol. 7, Humana Press, Clifton, NJ.Google Scholar
  15. Bourgain, R. H., Touqui, L., Maes, L., Braquet, P., Andries, R., Braquet, M., 1985, The effect of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-Acether) on the arterial wall, Prostaglandins, 30:185.PubMedGoogle Scholar
  16. Braquet, P., Borgeat, P., Etienne, A., and Braquet, M., 1985, Stimulus secretion coupling and leukotriene formation in the triggering of immediate hypersensitivity reactions, A. I. P. Immunol., 136:186.Google Scholar
  17. Buxton, D. B., Fisher, R. A., Hanahan, D. J., and Olson, M. S., 1986a, Platelet activating factor mediated vasoconstriction and glycogenolysis in the perfused rat liver, J. Biol. Chem., 261:644.PubMedGoogle Scholar
  18. Buxton, D. B., Hanahan, D. J., and Olson, M. S., 1986b, Specific antagonists of platelet activating factor mediated vasoconstriction and glycogenolysis in the perfused rat liver, Biochem. Pharmacol., 35:893.PubMedCrossRefGoogle Scholar
  19. Chen, S. F. and Chan, P. H., 1985, One step separation of free fatty acids and phospholipids in brain tissue extracts by high performance liquid chromatography, J. Chromatogr., 344:297.PubMedGoogle Scholar
  20. Chen, S. S. and Kou, A. Y., 1982, High-performance liquid chromatography of methylated phospholipids., J. Chromatogr., 232:237.PubMedGoogle Scholar
  21. Christie, W. W., 1985, Rapid separation and quantification of lipid classes by HPLC and mass (light scattering) detection, J. Lipid Res., 26:507.PubMedGoogle Scholar
  22. Colard, O., Breton, M., and Bereziat, G., 1986, Arachidonate mobilization in diacyl, alkylacyl and alkenylacyl phospholipids on stimulation of rat platelets by thrombin and the Ca2+ ionophore A23187, Biochem. J., 233:691.PubMedGoogle Scholar
  23. Creer, M. H. and Gross, R. W., 1985, Reversed phase high performance liquid Chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids, J. Chromatogr., 338:61.PubMedGoogle Scholar
  24. Demediuk, P., Saunders, R. D., Anderson, D. K., Means, E. D., and Horrocks, L. A., 1985, Membrane lipid changes in laminectomized and traumatized cat spinal cord, Proc. Nat. Acad. Sci. U.S.A., 82:7071.CrossRefGoogle Scholar
  25. Doyle, V. M., Creba, J. A., and Ruegg, U. T., 1986, Platelet activating factor mobilizes intracellular calcium in vascular smooth muscle cells, FEBS Lett., 197:13.PubMedCrossRefGoogle Scholar
  26. Dugan, L. L., Demediuk, P., Pendley II, C. E., and Horrocks, L. A., 1986a, Separation of phospholipids by HPLC: All major classes, including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine., J. Chromatog., 378:317.Google Scholar
  27. Dugan, L., Bazan, N. G., and Horrocks, L. A., 1986b, Extraction/HPLC separation of polyphosphoinositides by neutral solvents, Trans. Amer. Soc. Neurochem., 17:138.Google Scholar
  28. El Tamer, A., Record, M., Fouvel, J., Chap, H., and Douste-Blazy, L., 1984, Studies on ether phospholipids.1. A new method of determination using phospholipase-A1 from guinea-pig pancreas — Application to Krebs-II Ascites-cells, Biochim. Biophys. Acta, 793:213.PubMedCrossRefGoogle Scholar
  29. Feuerstein, G., Lux Jr., W. E., Ezra, D., Hayes, E. C., Snyder, F., and Faden, A. I., 1985, Thyrotropin-releasing hormone blocks the hypotensive effects of platelet-activating factor in the unanesthetized guinea pig, J. Cardiovasc. Pharmacol., 7:335.PubMedCrossRefGoogle Scholar
  30. Folch, J., Lees, M., and Sloane Stanley, G. H., 1957, A simple method for the isolation and purification of total lipides from animal tissues, J. Biol. Chem., 226:497.PubMedGoogle Scholar
  31. Gross, R. W., 1985, Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum, Biochemistry, 24:1662.PubMedCrossRefGoogle Scholar
  32. Gustavsson, L., 1986, Densitometric quantification of individual phospholipids. Improvement and evaluation of a method using molybdenum blue reagent for detection, J. Chromatogr., 375:255.PubMedGoogle Scholar
  33. Hanahan, D. J., 1986, Platelet activating factor: a biologically active phosphoglyceride, Annu. Rev. Biochem., 55:483.PubMedCrossRefGoogle Scholar
  34. Hara, A. and Radin, N. S., 1978, Lipid extraction of tissues with a low-toxicity solvent, Anal. Biochem., 90:420.PubMedCrossRefGoogle Scholar
  35. Hauser, G. and Eichberg, J., 1973, Improved conditions for the preservation and extraction of polyphosphoinositides, Biochim. Biophys. Acta, 326:201.PubMedCrossRefGoogle Scholar
  36. Hax, W. M. A., 1977, High-performance liquid Chromatographic separation and photometric detection of phospholipids, J. Chromatogr., 142:735.PubMedCrossRefGoogle Scholar
  37. Hayashi, H., Kudo, I., Inoue, K., Onozaki, K., Tsushima, S., Nomura, H., and Nojima, S., 1985, Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists, J. Biochem., 97:1737.PubMedGoogle Scholar
  38. Hirasawa, K. and Nishizuka, Y., 1985, Phosphatidylinositol turnover in receptors: mechanism and signal transduction, Ann. Rev. Pharmacol. Toxicol., 25:147.CrossRefGoogle Scholar
  39. Horrocks, L. A., 1968, The alk-l-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography, J. Lipid Res., 9:469.PubMedGoogle Scholar
  40. Horrocks, L. A., 1972, Content, composition, and metabolism of mammalian and avian lipids that contain ether groups, in: “Ether Lipids; Chemistry and Biology,” Snyder, F., ed., Academic Press, New York.Google Scholar
  41. Horrocks, L. A. and Sharma, M., 1982, “Phospholipids, New Comprehensive Biochemistry,” Hawthorne, J. N. and Ansell, G. B., eds., vol. 4, Elsevier Biomedical Press, Amsterdam.Google Scholar
  42. Horrocks, L. A. and Sun, G. Y., 1972, Ethanolamine plasmalogens, in: “Research Methods in Neurochemistry,” Rodnight, R. and Marks, N., eds., Plenum Press, New York.Google Scholar
  43. Horrocks, L. A., Harder, H. W., Mozzi, R., Goracci, G., Francescangeli, E., Porcellati, S., and Nenci, G. G., 1986a, “Enzymes of Lipid Metabolism,” Freysz, L. and Gatt, S., eds., vol. 2, Plenum Press, New York.Google Scholar
  44. Horrocks, L. A., Yeo, Y. Y., Harder, H. W., Mozzi, R., and Goracci, G., 1986b, Choline plasmalogens, glycerophospholipid methylation, and receptor-mediated activation of adenylate cyclase, Adv. Cyclic Nucleotide Protein Phosphorylation Res., 20:263.PubMedGoogle Scholar
  45. Hwang, S. B., Lam, M. H., Li, C. L., and Shen, T. Y., 1986, Release of platelet activating factor and its involvement in the 1st phase of carrageenin induced rat foot edema, Eur. J. Pharmacol., 120:33.PubMedCrossRefGoogle Scholar
  46. Kara, J., Brorvicka, M., Liebl, V., Smolikova, J., and Ubik, K., 1986, A novel nontoxic alkyl-phospholipid with selective antitumor activity, plasmanyl-(N-acyl)-ethanolamine (PNAE), isolated from degenerating chick embryonal tissues and from an anticancer biopreparation cACPL, Neoplasma, 33:198.Google Scholar
  47. Kinlough-Rathbone, J. D., Packham, M. A., and Mustard, J. F., 1983, Measurement of platelet function, in: “Methods in Haematology,” Harker, L. A. and Zimmerman, T. S., eds., Churchill-Livingstone, Edinburgh.Google Scholar
  48. Kito, M., Takamura, H., Narita, H., and Urade, R., 1985, A sensitive method for quantitative analysis of phospholipid molecular species by HPLC, J. Biochem., 98:327.PubMedGoogle Scholar
  49. Kostetskii, E. Y. and Sergeyuk, N. N., 1985, Phospholipids and their plasmalogen form in the muscle tissue of marine invertebrates, J. Evol. Biochem., 21:133.Google Scholar
  50. Lee, T. C., Malone, B., and Snyder, F., 1986, A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl CoA acetyltransferase in rat spleen., J. Biol. Chem., 261:5373.PubMedGoogle Scholar
  51. Loeb, L. A. and Gross, R. W., 1986, Identification and purification of sheep platelet phospholipase A2 isoforms, J. Biol. Chem., 261:10467.PubMedGoogle Scholar
  52. Malone, B., Lee, T. C., and Snyder, F., 1985, Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product, J. Biol. Chem., 260:1531.PubMedGoogle Scholar
  53. McKean, M. L. and Silver, M. J., 1985, Phospholipid biosynthesis in human platelets. The acylation of lyso platelet activating factor, Biochem. J., 225:723.PubMedGoogle Scholar
  54. Metz, S. A., 1986, Ether-linked lysophospholipids initiate insulin secretion. Lysophospholipids may mediate effects of phospholipase A2 activation on hormone release, Diabetes, 35:808.PubMedCrossRefGoogle Scholar
  55. Nakagawa, Y. and Horrocks, L. A., 1983, Separation of alkenylacyl, alkylacyl, and diacyl analogues and their molecular species by high performance liquid chromatography, J. Lipid Res., 24:1268.PubMedGoogle Scholar
  56. Natarajan, V., 1981, On the biosynthesis and metabolism of N-acylethanolamine phospholipids in infarcted dog heart, Biochim. Biophys. Acta, 774:445.Google Scholar
  57. Natarajan, V., Epps, D. E., Schmid, P. C., and Schmid, H. H. O., 1980, Accumulation of N-acylethanolamine glycerophospholipids in infarcted myocardium, Biochim. Biophys. Acta, 618:420.PubMedCrossRefGoogle Scholar
  58. Nishizuka, Y., 1984a, The role of protein kinase C in cell surface signal transduction and tumor promotion., Nature, 308:693.PubMedCrossRefGoogle Scholar
  59. Nishizuka, Y., 1984b, Turnover of inositol phospholipids and signal transduction., Science, 225:1365.PubMedCrossRefGoogle Scholar
  60. Oda, M., Satouchi, K., Yasunaga, K., and Saito, K., 1985, Production of platelet activating factor by washed rabbit platelets, J. Lipid Res., 26:1294.PubMedGoogle Scholar
  61. Orlov, S. M., Kulikov, V. I., Polner, A. A., and Bergelson, L. D., 1985, Release of histamine from human leukocytes induced by 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) and its structural analogs, Biochemistry-USSR, 50:575.Google Scholar
  62. Poitevin, B., Mencia-Huerta, J. M., Roubin, R., and Benveniste, J., 1985, Role of PAF acether (Platelet Activating Factor) in neutrophil activation, in: “Pulmonary Circulation and Acute Lung Injury,” Said, S. I., ed., Future Publishing Co.Google Scholar
  63. Porcellati, S., Goracci, G., Costantini, V., Pistolesi, R., Nenci, G. G., and Horrocks, L. A., 1986, October, Plasmalogens are metabolized in platelets during thrombin aggregation, Paper presented at Second International Conference, Platelet-Activating Factor and Structurally-Related Alkyl Ether Lipids, Gatlinburg, TN.Google Scholar
  64. Robinson, M. and Snyder, F., 1985, Metabolism of platelet activating factor by rat alveolar macrophages. Lyso PAF as an obligatory intermediate in the formation of alkylarachidonoyl glycerophosphocholine species, Biochim. Biophys. Acta, 837:52.PubMedCrossRefGoogle Scholar
  65. Rouser, G., Siakotos, A. N., and Fleischer, S., 1966, Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots, Lipids, 1:85.PubMedCrossRefGoogle Scholar
  66. Saeki, S., Musugi, F., Ogihara, T., Otsuka, A., Kumahara, Y., Watanabe, K., Tamura, K., Akashi, A., and Kumagai, A., 1985, Effects of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (Platelet activating factor) on cardiac function in perfused guinea pig heart, Life Sci., 37:325.PubMedCrossRefGoogle Scholar
  67. Sanchez-Crespo, M., Alonso, F., Garcia-Gil, M., Gomez-Cambrone, J., and Neito, M. L., 1985, Synthesis of platelet activating factor from human polymorphonuclear leukocytes. Regulation and pharmacological approaches, Int. J. Tissue Res., 7:345.Google Scholar
  68. Saunders, R. D. and Horrocks, L. A., 1984, Simultaneous extraction and preparation for high performance liquid chromatography of prostaglandins and phospholipids, Anal. Biochem., 143:71.PubMedCrossRefGoogle Scholar
  69. Snyder, F., 1985, Chemical and biochemical aspects of platelet activating factor: a novel class of acetylated ether-linked choline phospholipids, Med. Res. Rev., 5:107.PubMedCrossRefGoogle Scholar
  70. Stokes, B. T., Fox, P., and Hollinden, G., 1983, Extracellular calcium activity in the injured spinal cord, Exp. Neurol., 80:561.PubMedCrossRefGoogle Scholar
  71. Stokes, B. T., Fox, P., and Hollinden, G., 1985, Extracellular metabolites: Their measurement and role in the acute phase of spinal cord injury, in: “Trauma of the Central Nervous System,” Dacey, J. R. G. E. A., ed., Raven Press, New York.Google Scholar
  72. Stokes, B. T., Hollinden, G., and Fox, P., 1984, Improvement in injury induced hypocalcia by high-dose naloxone intervention, Brain Res., 290:187.PubMedCrossRefGoogle Scholar
  73. Sugiura, T. and Waku, K., 1985, CoA independent transfer of arachidonic acid from 1,2-diacyl-sn-glycero-3-phosphocholine to 1-0-alkyl-sn-glycero-3-phosphocholine (lyso platelet-activating factor) by macrophage microsomes, Biochem. Biophys. Res. Commun., 127:384.PubMedCrossRefGoogle Scholar
  74. Sugiura, T., Masuzawa, Y., and Waku, K., 1985, Transacylation of 1-0-alkyl-sn-glycero-3-phosphocholine (lyso platelet-activating factor) and 1-0-alkenyl-sn-glycero-3-phosphoethanolamine with docosahexaenoic acid (22:6ω3), Biochem. Biophys. Res. Commun., 133:574.PubMedCrossRefGoogle Scholar
  75. Touqui, L., Jacquemin, C., Dumarey, C., and Vargaftig, B. B., 1985, 1-0-Alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl glycerophosphorylcholine cycle, Biochim. Biophys. Acta, 833:111.PubMedCrossRefGoogle Scholar
  76. Venuti, M. C., 1985, Platelet activating factor. Multifaceted biochemical and physiological mediator, Annu. Rep. Med. Chem., 20:193.CrossRefGoogle Scholar
  77. Wolf, R. A. and Gross, R. W., 1985, Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium, J. Biol. Chem., 260:7295.PubMedGoogle Scholar
  78. Young, W. and Flamm, E. S., 1982, Effect of high-dose corticosteroid therapy on blood flow, evoked potentials, and extracellular calcium in experimental spinal injury, J. Neurosurg., 57:667.PubMedCrossRefGoogle Scholar
  79. Young, W. and Koreh, I., 1986, Potassium and calcium changes in injured spinal cords, Brain Res., 365:42.PubMedCrossRefGoogle Scholar
  80. Yousufzai, S. Y. and Abdel-Latif, A. A., 1985, Effects of platelet activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle, Fed. Proc, 44:488.Google Scholar

Copyright information

© Springer Science+Business Media New York 1987

Authors and Affiliations

  • Lloyd A. Horrocks
    • 1
  • Laura L. Dugan
    • 1
  • Cheryl J. Flynn
    • 1
  • Gianfrancesco Goracci
    • 1
  • Serena Porcellati
    • 1
  • Young Yeo
    • 1
  1. 1.Department of Physiological ChemistryThe Ohio State UniversityColumbusUSA

Personalised recommendations