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Simultaneous Analysis of Multiple Lipid Oxidation Products In Vivo by Liquid Chromatographic-Mass Spectrometry (LC-MS)

  • Huiyong Yin
  • Todd Davis
  • Ned A. Porter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

Free radical-induced oxidation of polyunsaturated fatty acid (PUFAs) has been linked to a number of human diseases including atherosclerosis and neurodegenerative disorders. Oxidation of PUFAs generates hydroperoxides and cyclic peroxides that are reduced to lipid alcohol, such as hydroxyeicosatetraenoic acid (HETEs), and isoprostanes (IsoPs) respectively. The IsoPs are isomers of prostaglandins that are generated from autoxidation of arachidonic acid (C20:4). Quantification of F2-IsoPs has been regarded as the “gold standard” to assess oxidative stress status in various human diseases. We herein report the protocol of analyzing HETEs and F2-IsoPs using a triple quadrupole mass spectrometer coupled to reverse phase liquid chromatography. The selected reaction monitoring (SRM) mode selects the parent ion of interest in the first Quad (m/z 319 for HETE and m/z 353 for F2-IsoPs) and fragments it in the second while an ion characteristic of the analyte of interest is monitored in the third Quad. This highly selective technique permits the simultaneous analysis of multiple oxidation products such as the HETEs and F2-IsoPs. This LC-MS technique can be applied to study the free radical oxidation mechanism in vitro and assess the oxidative stress status in biological tissues and fluids.

Key words

Lipid peroxidation free radicals liquid chromatography – mass spectrometry (LC-MS) isoprostanes oxidative stress biomarkers 

Abbreviation

APCI

atmospheric pressure chemical ionization

BHT

butylated hydroxytoluene

CID

collision induced dissociation

ESI-MS

electrospray mass spectrometry

GC

gas chromatography

HPLC

high performance liquid chromatography

IsoP

isoprostane

IPA

isopropanol

LC

liquid chromatography

MS

mass spectrometry

PG

prostaglandin

PGF2α

Prostaglandin F2 α

HpETE

hydroperoxyeicosatetraenoate

SRM

selective reaction monitoring.

Notes

Acknowledgments

We thank Dr. David Hachey, Ms. Lisa Mannier, and Mrs. Betty Fox of the Mass Spectrometry Research Center of Vanderbilt University for assistance with the MS analysis. We acknowledge discussion with Dr. Jason Morrow in the Division of Clinical Pharmacology at Vanderbilt University. Financial support from NIEHS P01 ES013125, NIH grants DK 48831, RR00095, CA 77839, HL17921, GM15431, P30 ES00267 (HY, pilot grant), and NSF CHE 0412493 is gratefully acknowledged.

References

  1. 1.
    Funk, C.D. (2001) Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science 294, 1871–1875.CrossRefPubMedGoogle Scholar
  2. 2.
    Chisolm, G.M. and Steinberg, D. (2000) The oxidative modification hypothesis of atherosclerosis: An Overview. Free Radic. Biol. Med. 28, 1815–1826.CrossRefPubMedGoogle Scholar
  3. 3.
    Montine, K.S., Quinn, J.F., Zhang, J., Fessel, J.P., Roberts, L.J., Jr., Morrow, J.D., and Montine, T.J. (2004) Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem. Phys. Lipids 128, 117–124.CrossRefPubMedGoogle Scholar
  4. 4.
    Montine, T.J., Markesberry, W.R., Morrow, J.D., and Roberts, L.J., Jr. (1998) Cerebrospinal fluid F-2-isoprostane levels are increased in Alzheimer’s disease. Annals Neurol. 44, 410–413.CrossRefGoogle Scholar
  5. 5.
    Kikugawa, K. (1997) Use and limitation of thiobarbituric acid (TBA) test for lipid peroxidation. Recent Res. Devel. Lipids Res. 1, 73–96.Google Scholar
  6. 6.
    Kenar, J.A., Havrilla, C.M., Porter, N.A., Guyton, J.R., Brown, S.A., Klemp, K.R., and Selinger, E. (1996) Identification and quantification of the regioisomeric cholesteryl linoleate hydroperoxides in oxidized human low density lipoprotein and high density lipoprotein. Chem. Res. Toxicol. 9, 737–744.CrossRefPubMedGoogle Scholar
  7. 7.
    Jessup, W., Dean, R.T., and Gebicki, J.M. (1994) Iodometric determination of hydroperoxides in lipids and proteins. Methods Enzymol. 233, 289–320.CrossRefGoogle Scholar
  8. 8.
    Basu, S. (2004) Isoprostanes: Novel bioactive products of lipid peroxidation. Free Radic. Res. 38, 102–105.CrossRefGoogle Scholar
  9. 9.
    Halliwell, B.G. (1999) Free Radicals in Biology and Medicine, 3rd Edition, Oxford University Press, NY.Google Scholar
  10. 10.
    Morrow, J.D., Hill, E., Burk, R.F., Nammour, T.M., Badr, K.F., and Roberts, L.J., Jr. (1990) A series of prostaglandin-F2-like compounds are produced in vivo in humans by a noncyclooxygenase, Free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87, 9383–9387.CrossRefPubMedGoogle Scholar
  11. 11.
    Porter, N.A. (1986) Mechanisms for the autoxidation of polyunsaturated lipids. Acc. Chem. Res. 19, 262–268.CrossRefGoogle Scholar
  12. 12.
    Porter, N.A., Caldwell, S.E., and Mills, K.A. (1995) Mechanisms of free radical oxidation of unsaturated lipids. Lipids 30, 277–290.CrossRefPubMedGoogle Scholar
  13. 13.
    Pryor, W.A. and Stanley, J.P. (1975) Suggested mechanism for the production of malonaldehyde during the autoxidation of polyunsaturated fatty acids. Nonenzymic production of prostaglandin endoperoxides during autoxidation. J. Org. Chem. 40, 3615–3617; see also Porter, N.A. and Funk, M.O. (1975) Peroxy radical cyclization as a model for prostaglandin biosynthesis. J. Org. Chem. 40, 3614–3615.CrossRefPubMedGoogle Scholar
  14. 14.
    Roberts, L.J., Jr. and Morrow, J.D. (2000) Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Radic. Biol. Med. 28, 505–513.CrossRefPubMedGoogle Scholar
  15. 15.
    Morrow, J.D., Minton, T.A., and Roberts, L.J.I. (1992) The F2-isoprostane, 8-epi-prostaglandin F2a, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins 42, 155–163.CrossRefGoogle Scholar
  16. 16.
    Pratico, D., Smyth, E.M., Violi, F., and FitzGerald, G.A. (1996) Local amplification of platelet function by 8-epi prostaglandin F is not mediated by thromboxane receptor isoforms. J. Biol. Chem. 271, 14916–14924.CrossRefPubMedGoogle Scholar
  17. 17.
    Kunapuli, P., Lawson, J.A., Rokach, J.A., Meinkoth, J.L., and FitzGerald, G.A. (1998) Prostaglandin F (PGF) and the isoprostane, 8,12-iso-isoprostane F2alpha -III, induce cardiomyocyte hypertrophy. Differential activation of downstream signaling pathways. J. Biol. Chem. 273, 22442–22452.CrossRefPubMedGoogle Scholar
  18. 18.
    Pratico, D., Lawson, J.A., and FitzGerald, G.A. (1995) Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F2. J. Biol. Chem. 270, 9800–9808.CrossRefPubMedGoogle Scholar
  19. 19.
    Yin, H., and Porter, N.A. (2005) New insights regarding the autoxidation of polyunsaturated fatty acids. Antiox. Redox Signal 7, 170–184.CrossRefGoogle Scholar
  20. 20.
    Yin, H., Havrilla, C.M., Morrow, J.D., and Porter, N.A. (2002) Formation of isoprostane bicyclic endoperoxides from the autoxidation of cholesteryl arachidonate. J. Am. Chem. Soc. 124, 7745–7754.CrossRefPubMedGoogle Scholar
  21. 21.
    Yin, H., Havrilla, C.M., Gao, L., Morrow, J.D., and Porter, N.A. (2003) Mechanisms for the formation of isoprostane endoperoxides from arachidonic acid: “Dioxetane” intermediate or beta-fragmentation of peroxyl radicals? J. Biol. Chem. 278, 16720–16725.CrossRefPubMedGoogle Scholar
  22. 22.
    Lawson, J.A., Rokach, J., and FitzGerald, G.A. (1999) Isoprostanes: Formation, analysis and use as indices of lipid peroxidation in vivo. J. Biol. Chem. 274, 24441–24444.CrossRefPubMedGoogle Scholar
  23. 23.
    Li, H., Lawson, J.A., Reilly, M., Adiyaman, M., Hwang, S.-W., Rokach, J., and FitzGerald, G.A. (1999) Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine. Proc. Natl. Acad. Sci. USA 96, 13381–13386.CrossRefPubMedGoogle Scholar
  24. 24.
    Kuhn, H. and O’Donnell, V.B. (2006) Inflammation and immune regulation by 12/15-lipoxygenases. Progr. Lipid Res. 45, 334–356.CrossRefGoogle Scholar
  25. 25.
    Schneider, C., Boeglin, W.E., and Brash, A.R. (2000) Enantiomeric separation of hydroxy eicosanoids by chiral column chromatography: Effect of the alcohol modifier. Anal. Biochem. 287, 186–189.CrossRefPubMedGoogle Scholar
  26. 26.
    Yin, H., Morrow, J.D., and Porter, N.A. (2004) Identification of a novel class of endoperoxides from arachidonate autoxidation. J. Biol. Chem. 279, 3766–3776.CrossRefPubMedGoogle Scholar
  27. 27.
    Yin, H., Porter, N.A., and Morrow, J.D. (2005) Separation and identification of F2-isoprostane regioisomers and diastereomers by novel liquid chromatographic/mass spectrometric methods. J. Chromatogr. B 827, 157–164.CrossRefGoogle Scholar
  28. 28.
    Porter, N.A., Logan, J., and Kontoyiannidou, V. (1979) Preparation and purification of arachidonic acid hydroperoxides of biological importance. J. Org. Chem. 44, 3177–3181.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Huiyong Yin
    • 1
  • Todd Davis
    • 2
  • Ned A. Porter
    • 3
  1. 1.Division of Clinical PharmacologyDepartments of Medicine and Chemistry Vanderbilt UniversityNashvilleUSA
  2. 2.Department of ChemistryIdaho State UniversityPocatelloUSA
  3. 3.Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt Institute of Chemical BiologyVanderbilt UniversityNashvilleUSA

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