Lipidomics pp 513-535 | Cite as

Identifying the Spatial Distribution of Vitamin E, Pulmonary Surfactant and Membrane Lipids in Cells and Tissue by Confocal Raman Microscopy

  • J. Renwick Beattie
  • Bettina C. Schock
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 579)

Summary

Every organ compromises of several different cell types. When studying the effects of a chosen compound within this organ or tissue uptake, localisation, metabolism, and the effect itself can be expected to differ between cells. Using the example of Vitamin E in pulmonary tissue we introduce confocal Raman Microscopy as a superior method to localise lipid-soluble compounds within tissues and cells. We describe the analyses of vitamin E, its oxidation products, and metabolites as well as pulmonary surfactant phospholipids in fixed lung tissue sections. Examples of main structural membrane lipids (PC, cholesterol) and an example of a lipid-signalling molecule (ceramide) are also included. Confocal Raman microscopy is a non-destructive optical method of analysing chemical and physical composition of solids, liquids, gases, gels, and solutions. The method is rich in information allowing discrimination of chemically similar molecules (including geometric isomers) and sensitive monitoring of subtle physical interactions. Additionally, Raman spectroscopy is relatively insensitive to water allowing the analysis of aqueous solutions and suspensions typical in biochemistry. In contrast, Raman spectroscopy is sensitive to non-polar molecules making it ideal for lipidomics research.

Key words

Raman Microscopy Cell Imaging Membrane Lipids Vitamin E Surfactant lipids Subcellular lipid distribution Lipid metabolites Lipid signalling 

Notes

Acknowledgement

Research was supported by a grant from the BBSRC, UK (JREI 18471) and the R&D Office, Northern Ireland (SPI/2384/03). JRB is supported by a grant from the Medical Research Council (MRC), UK (G0600053). The authors thank Mr. C. Maguire for technical support. BLES™ was a gift from BLES Biochemicals Inc., Canada (H. Dick) and the tocopherol metabolite was a gift from F. Galli and F. Mazzini (University of Perugia, Italy). The authors are very grateful for these contributions.

References

  1. 1.
    Beattie, J.R., Maguire, C., Gilchrist, S., Barrett, L.J., Cross, C.E., Possmayer, F., Ennis, M., Elborn, J.S., Curry, W.J., McGarvey, J.J., and Schock, B.C.,(2007) The use of Raman microscopy to determine and localize vitamin E in biological samples. FASEB J. 21, p. 766–76.PubMedCrossRefGoogle Scholar
  2. 2.
    Kolleck, I., Schlame, M., Fechner, H., Looman, A.C., Wissel, H., and Rustow, B.,(1999) HDL is the major source of vitamin E for type II pneumocytes. Free Radic Biol Med. 27, p. 882–90.PubMedCrossRefGoogle Scholar
  3. 3.
    Hosomi, A., Goto, K., Kondo, H., Iwatsubo, T., Yokota, T., Ogawa, M., Arita, M., Aoki, J., Arai, H., and Inoue, K.,(1998) Localization of alpha-tocopherol transfer protein in rat brain. Neurosci Lett. 256, p. 159–62.PubMedCrossRefGoogle Scholar
  4. 4.
    You, C.S., Sontag, T.J., Swanson, J.E., and Parker, R.S.,(2005) Long-chain carboxychromanols are the major metabolites of tocopherols and tocotrienols in A549 lung epithelial cells but not HepG2 cells. J Nutr. 135, p. 227–32.PubMedGoogle Scholar
  5. 5.
    Jiang, Q., Freiser, H., Wood, K.V., and Yin, X.,(2007) Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats. J Lipid Res. 48, p. 1221–30.PubMedCrossRefGoogle Scholar
  6. 6.
    Hac-Wydro, K., Kapusta, J., Jagoda, A., Wydro, P., and Dynarowicz-Latka, P.,(2007) The influence of phospholipid structure on the interactions with nystatin, a polyene antifungal antibiotic A Langmuir monolayer study. Chem Phys Lipids. 150, p. 125–35.PubMedCrossRefGoogle Scholar
  7. 7.
    Galli, F., Lee, R., Dunster, C., and Kelly, F.J.,(2002) Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma. Free Radic Biol Med. 32, p. 333–40.PubMedCrossRefGoogle Scholar
  8. 8.
    Leonard, S.W., Paterson, E., Atkinson, J.K., Ramakrishnan, R., Cross, C.E., and Traber, M.G.,(2005) Studies in humans using deuterium-labeled alpha- and gamma-tocopherols demonstrate faster plasma gamma-tocopherol disappearance and greater gamma-metabolite production. Free Radic Biol Med. 38, p. 857–866.PubMedCrossRefGoogle Scholar
  9. 9.
    Hybertson, B.M., Chung, J.H., Fini, M.A., Lee, Y.M., Allard, J.D., Hansen, B.N., Cho, O.J., Shibao, G.N., and Repine, J.E.,(2005) Aerosol-administered alpha-tocopherol attenuates lung inflammation in rats given lipopolysaccharide intratracheally. Exp Lung Res. 31, p. 283–94.PubMedCrossRefGoogle Scholar
  10. 10.
    Morita, N., Traber, M.G., Enkhbaatar, P., Westphal, M., Murakami, K., Leonard, S.W., Cox, R.A., Hawkins, H.K., Herndon, D., Traber, L.D., and Traber, D.L.,(2006) Aerosolized alpha-tocopherol ameliorates acute lung injury following combined burn and smoke inhalation injury in sheep. Shock. 25, p. 277–82.PubMedCrossRefGoogle Scholar
  11. 11.
    Enkhbaatar, P., Cox, R.A., Traber, L.D., Westphal, M., Aimalohi, E., Morita, N., Prough, D.S., Herndon, D.N., and Traber, D.L.,(2007) Aerosolized anticoagulants ameliorate acute lung injury in sheep after exposure to burn and smoke inhalation. Crit Care Med. 12, p. 2805–10CrossRefGoogle Scholar
  12. 12.
    Palmieri, T.L., Enkhbaatar, P., Bayliss, R., Traber, L.D., Cox, R.A., Hawkins, H.K., Herndon, D.N., Greenhalgh, D.G., and Traber, D.L.,(2006) Continuous nebulized albuterol attenuates acute lung injury in an ovine model of combined burn and smoke inhalation. Crit Care Med. 34, p. 1719–24.PubMedCrossRefGoogle Scholar
  13. 13.
    van Meer, G., Voelker, D.R., and Feigenson, G.W.,(2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol. 9, p. 112–24.PubMedCrossRefGoogle Scholar
  14. 14.
    Marsh, D.,(2007) Lateral pressure profile, spontaneous curvature frustration, and the incorporation and conformation of proteins in membranes. Biophys J. 93, p. 3884–99.PubMedCrossRefGoogle Scholar
  15. 15.
    Hannun, Y.A. and Obeid, L.M.,(2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol. 9, p. 139–50.PubMedCrossRefGoogle Scholar
  16. 16.
    Wu, D. and Meydani, S.N.,(2008) Age-associated changes in immune and inflammatory responses: impact of vitamin E intervention. J Leukoc Biol. 84, p. 900–14PubMedCrossRefGoogle Scholar
  17. 17.
    Teichgraber, V., Ulrich, M., Endlich, N., Riethmuller, J., Wilker, B., De Oliveira-Munding, C.C., van Heeckeren, A.M., Barr, M.L., von Kurthy, G., Schmid, K.W., Weller, M., Tummler, B., Lang, F., Grassme, H., Doring, G., and Gulbins, E.,(2008) Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med. 14, p. 382–91.PubMedCrossRefGoogle Scholar
  18. 18.
    Jobe, A.H. and Ikegami, M.,(2001) Biology of surfactant. Clin Perinatol. 28, p. 655–69, vii–viii.PubMedCrossRefGoogle Scholar
  19. 19.
    Orgeig, S., Daniels, C.B., Johnston, S.D., and Sullivan, L.C.,(2003) The pattern of surfactant cholesterol during vertebrate evolution and development: does ontogeny recapitulate phylogeny? Reprod Fertil Dev. 15, p. 55–73.PubMedCrossRefGoogle Scholar
  20. 20.
    Panda, A.K., Nag, K., Harbottle, R.R., Rodriguez-Capote, K., Veldhuizen, R.A., Petersen, N.O., and Possmayer, F.,(2004) Effect of acute lung injury on structure and function of pulmonary surfactant films. Am J Respir Cell Mol Biol. 30, p. 641–50.PubMedCrossRefGoogle Scholar
  21. 21.
    Gunasekara, L., Schurch, S., Schoel, W.M., Nag, K., Leonenko, Z., Haufs, M., and Amrein, M.,(2005) Pulmonary surfactant function is abolished by an elevated proportion of cholesterol. Biochim Biophys Acta. 1737, p. 27–35.PubMedCrossRefGoogle Scholar
  22. 22.
    Ingold, K.U., Webb, A.C., Witter, D., Burton, G.W., Metcalfe, T.A., and Muller, D.P.,(1987) Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency. Arch Biochem Biophys. 259, p. 224–5.PubMedCrossRefGoogle Scholar
  23. 23.
    Traber, M.G., Frei, B., and Beckman, J.S.,(2008) Vitamin E revisited: do new data validate benefits for chronic disease prevention? Curr Opin Lipidol. 19, p. 30–8.PubMedGoogle Scholar
  24. 24.
    Traber, M.G.,(2005) Vitamin E, in Modern Nutrition in Health and Disease, (Shils, M.E., Olson, J.A., Shike, M., and Ross, A.C., eds.), Lippincott Williams & Wilkins: Baltimore. p. 396–411.Google Scholar
  25. 25.
    Cross, C.E., van der Vliet, A., O’Neill, C.A., Louie, S., and Halliwell, B.,(1994) Oxidants, antioxidants, and respiratory tract lining fluids. Environ Health Perspect. 102 Suppl 10, p. 185–91.PubMedCrossRefGoogle Scholar
  26. 26.
    Bouhafs, R.K. and Jarstrand, C.,(1999) Phagocyte-induced lipid peroxidation of lung surfactant. Pediatr Pulmonol. 27, p. 322–7.PubMedCrossRefGoogle Scholar
  27. 27.
    Bouhafs, R.K. and Jarstrand, C.,(1999) Lipid peroxidation of lung surfactant by bacteria. Lung. 177, p. 101–10.PubMedCrossRefGoogle Scholar
  28. 28.
    Atkinson, J., Epand, R.F., and Epand, R.M.,(2008) Tocopherols and tocotrienols in membranes: a critical review. Free Radic Biol Med. 44, p. 739–64.PubMedCrossRefGoogle Scholar
  29. 29.
    Cuschieri, J., Bulger, E., Biligren, J., Garcia, I., and Maier, R.V.,(2007) Vitamin E inhibits endotoxin-mediated transport of phosphatases to lipid rafts. Shock. 27, p. 19–24.PubMedCrossRefGoogle Scholar
  30. 30.
    Barbas, C., Castro, M., Bonet, B., Viana, M., and Herrera, E.,(1997) Simultaneous determination of vitamins A and E in rat tissues by high-performance liquid chromatography. J Chromatogr A. 778, p. 415–20.PubMedCrossRefGoogle Scholar
  31. 31.
    Ito, Y., Ochiai, J., Sasaki, R., Suzuki, S., Kusuhara, Y., Morimitsu, Y., Otani, M., and Aoki, K.,(1990) Serum concentrations of carotenoids, retinol, and alpha-tocopherol in healthy persons determined by high-performance liquid chromatography. Clin Chim Acta. 194, p. 131–44.PubMedCrossRefGoogle Scholar
  32. 32.
    Podda, M., Weber, C., Traber, M.G., and Packer, L.,(1996) Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols, and ubiquinones. J Lipid Res. 37, p. 893–901.PubMedGoogle Scholar
  33. 33.
    Ikenoya, S., Abe, K., Tsuda, T., Yamano, Y., Hiroshima, O., Ohmae, M., and Kawabe, K.,(1979) Electrochemical detector for high-performance liquid chromatography. II. Determination of tocopherols, ubiquinones and phylloquinone in blood. Chem Pharm Bull (Tokyo). 27, p. 1237–44.CrossRefGoogle Scholar
  34. 34.
    Leonard, S.W., Gumpricht, E., Devereaux, M.W., Sokol, R.J., and Traber, M.G.,(2005) Quantitation of rat liver vitamin E metabolites by LC–MS during high-dose vitamin E administration. J. Lipid. Res. 46, p. 1068–75.PubMedCrossRefGoogle Scholar
  35. 35.
    Leonard, S.W., Bruno, R.S., Paterson, E., Schock, B.C., Atkinson, J., Bray, T.M., Cross, C.E., and Traber, M.G.,(2003) 5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo. Free Radic Biol Med. 35, p. 1560–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Galli, F., Lee, R., Dunster, C., and Kelly, F.J.,(2002) Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma. Free Radic Biol Med. 32, p. 333–40.PubMedCrossRefGoogle Scholar
  37. 37.
    Saito, Y., Yoshida, Y., Nishio, K., Hayakawa, M., and Niki, E.,(2004) Characterization of cellular uptake and distribution of vitamin E. Ann N Y Acad Sci. 1031, p. 368–75.PubMedCrossRefGoogle Scholar
  38. 38.
    Panin, L.E., Polyakov, L.M., Kolosova, N.G., Russkikh, G.S., and Poteryaeva, O.N.,(1998) Distribution of tocopherol and apolipoprotein A-I immunoreactivity in rat liver chromatin. Membr Cell Biol. 11, p. 631–40.PubMedGoogle Scholar
  39. 39.
    Kiss, E., Nagy, P., Balogh, A., Szollosi, J., and Matko, J.,(2008) Cytometry of raft and caveola membrane microdomains: from flow and imaging techniques to high throughput screening assays. Cytometry A. 73, p. 599–614.PubMedGoogle Scholar
  40. 40.
    Gombos, I., Steinbach, G., Pomozi, I., Balogh, A., Vamosi, G., Gansen, A., Laszlo, G., Garab, G., and Matko, J.,(2008) Some new faces of membrane microdomains: a complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells. Cytometry A. 73, p. 220–9.PubMedGoogle Scholar
  41. 41.
    Biro, A., Cervenak, L., Balogh, A., Lorincz, A., Uray, K., Horvath, A., Romics, L., Matko, J., Fust, G., and Laszlo, G.,(2007) Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions. J Lipid Res. 48, p. 19–29.PubMedCrossRefGoogle Scholar
  42. 42.
    Touboul, D., Brunelle, A., Halgand, F., De La Porte, S., and Laprevote, O.,(2005) Lipid imaging by gold cluster time-of-flight secondary ion mass spectrometry: application to Duchenne muscular dystrophy. J Lipid Res. 46, p. 1388–95.PubMedCrossRefGoogle Scholar
  43. 43.
    Beattie, J.R., Bell, S.E.J., Borgaard, C., Fearon, A., and Moss, B.W.,(2006) Prediction of adipose tissue composition using Raman spectroscopy: Average properties and individual fatty acids. Lipids. 41, p. 287–294.PubMedCrossRefGoogle Scholar
  44. 44.
    Beattie, J.R., Bell, S.E.J., Borgaard, C., Fearon, A.M., and Moss, B.W.,(2004) Multivariate prediction of clarified butter composition using Raman spectroscopy. Lipids. 39, p. 897–906.PubMedCrossRefGoogle Scholar
  45. 45.
    Hancewicz, T.M. and Petty, C.,(1995) Quantitative analysis of vitamin A using Fourier transform Raman spectroscopy. Spectroc Acta Pt A-Molec Biomolec Spectr. 51, p. 2193–2198.CrossRefGoogle Scholar
  46. 46.
    Schulz, H. and Baranska, M.,(2007) Identification and quantification of valuable plant substances by IR and Raman spectroscopy. Vib Spectrosc. 43, p. 12–25.CrossRefGoogle Scholar
  47. 47.
    Beattie, J.R., Bell, S.E., and Moss, B.W.,(2004) A critical evaluation of Raman spectroscopy for the analysis of lipids: fatty acid methyl esters. Lipids. 39, p. 407–19.PubMedCrossRefGoogle Scholar
  48. 48.
    Bunow, M.R. and Levin, I.R.,(1977) Raman spectra and vibrational assignments for deuterated membrane lipids. 1,2 dipalmitoyl phoshatisylcholine-d9 and d62. Biochim Biophys Acta. 489, p. 191–206.PubMedCrossRefGoogle Scholar
  49. 49.
    Kint, S., Wermer, P.H., and Scherer, J.R.,(1992) Raman-Spectra of hydrated phospholipid-bilayers 2. Water and headgroup interactions. J Phys Chem. 96, p. 446–452.CrossRefGoogle Scholar
  50. 50.
    Lawson, E.E., Anigbogu, A.N.C., Williams, A.C., Barry, B.W., and Edwards, H.G.M.,(1998) Thermally induced molecular disorder in human stratum corneum lipids compared with a model phospholipid system; ft-Raman spectroscopy. Spectrochim Acta A-Mol Biomol Spectros. 54, p. 543–558.CrossRefGoogle Scholar
  51. 51.
    Wallach, D.F.H., Verma, S.P., and Fookson, J.,(1979) Application of laser Raman and infrared spectroscopy to the analysis of membrane structure. Biochim Biophys Acta. 559, p. 153–208.PubMedCrossRefGoogle Scholar
  52. 52.
    Beattie, J.R., Brockbank, S., McGarvey, J.J., and Curry, W.J.,(2005) Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis. Mol Vis. 11, p. 825–832.PubMedGoogle Scholar
  53. 53.
    Haka, A.S., Volynskaya, Z., Gardecki, J.A., Nazemi, J., Lyons, J., Hicks, D., Fitzmaurice, M., Dasari, R.R., Crowe, J.P., and Feld, M.S.,(2006) In vivo margin assessment during partial mastectomy breast surgery using Raman spectroscopy. Cancer Res. 66, p. 3317–3322.PubMedCrossRefGoogle Scholar
  54. 54.
    Min, Y.K., Yamamoto, T., Kohda, E., Ito, T., and Hamaguchi, H.,(2005) 1064 nm near-infrared multichannel Raman spectroscopy of fresh human lung tissues. J Raman Spectrosc. 36, p. 73–76.CrossRefGoogle Scholar
  55. 55.
    Lajoie, P. and Nabi, I.R., (2007) Regulation of raft-dependent endocytosis. J Cell Mol Med. 11, p. 644–53.PubMedCrossRefGoogle Scholar
  56. 56.
    Eriksson, L., Johansson, E., Kettaneh-Wold, N., Trygg, J., Wikström, C., and Wold, S., eds. Multi- and Megavariate Data Analysis Part I: Basic Principles and Applications, Second revised and enlarged edition 2nd ed. Vol. 1. 2006, Umetrics: Umea. p. 425.Google Scholar
  57. 57.
    Eriksson, L., Johansson, E., Kettaneh-Wold, N., Trygg, J., Wikström, C., and Wold, S., eds. Multi- and Megavariate Data Analysis Part II: Advanced Applications and Method Extensions, Second revised and enlarged edition 2nd ed. Vol. 2. 2006, Umetrics: Umea. 345.Google Scholar
  58. 58.
    McCreery, R.L.,(2000) Raman Spectroscopy for Chemical Analysis. 1 ed. in Chemical Analysis: a Series of Monographs of Analytical Chemistry and its Applications., (Winefordner, J.D., ed). Wiley, New York.Google Scholar
  59. 59.
    Tu, A.T.,(1982) Raman Spectroscopy in Biology: Principles and Applications. Wiley, New York.Google Scholar
  60. 60.
    Bell, S.B., JR; McGarvey, J.J.; Peters, K L., Sirimuthu, N.M.S., Speers, S J.,(2004) Development of sampling methods for Raman analysis of solid dosage forms of therapeutic and illicit drugs. J Raman Spectrosc. 35, p. 409–417.CrossRefGoogle Scholar
  61. 61.
    Glenn, J.V., Beattie, J.R., Barrett, L., Frizzell, N., Thorpe, S.R., Boulton, M.E., McGarvey, J.J., and Stitt, A.W.,(2007) Confocal Raman microscopy can quantify advanced glycation end product (AGE) modifications in Bruch’s membrane leading to accurate, nondestructive prediction of ocular aging. FASEB J. 21, p. 3542–52.PubMedCrossRefGoogle Scholar
  62. 62.
    Beattie, J.R., Glenn, J.V., Boulton, M.E., Stitt, A.W., and McGarvey, J.J.,(2009) Effect of signal intensity normalization on the multivariate analysis of spectral data in complex ‘real-world’ datasets. J Raman Spectrosc. 40, p. 429–435.CrossRefGoogle Scholar
  63. 63.
    Jirasek, A., Schulze, G., Yu, M.M.L., Blades, W., and Turner, R.F.B.,(2004) Accuracy and precision of manual baseline determination. Appl Spectrosc. 58, p. 1488–1499.PubMedCrossRefGoogle Scholar
  64. 64.
    Lieber, C.A. and Mahadevan-Jansen, A.,(2003) Automated method for subtraction of fluorescence from biological Raman spectra. Appl Spectrosc. 57, p. 1363–1367.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • J. Renwick Beattie
    • 1
  • Bettina C. Schock
    • 2
  1. 1.School of Medicine, Dentistry and Biomedical SciencesQueen’s UniversityBelfastUK
  2. 2.School of Medicine, Dentistry and Biomedical SciencesQueen’s University of BelfastBelfastUK

Personalised recommendations