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Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin γ-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry

Nature Protocols volume 2, pages 2079–2091 (2007)Cite this article

Abstract

Measurement of F2-isoprostanes (F2-IsoPs) has been independently verified as one of the most reliable approaches to assess oxidative stress in vivo. However, the rapid clearance of F2-IsoPs makes the timing of sample collection critical for short-lived oxidative insults. Isoketals (IsoKs) are γ-ketoaldehydes formed via the IsoP pathway of lipid peroxidation that rapidly react with lysyl residues of proteins to form stable protein adducts. Oxidative stress can also activate cyclooxygenases to produce prostaglandin H2, which can form two specific isomers of IsoK—levuglandin (LG) D2 and E2. Because adducted proteins are not rapidly cleared, IsoK/LG protein adduct levels can serve as a dosimeter of oxidative and inflammatory damage over prolonged periods of time as well as brief episodes of injury. Quantification of IsoK/LG protein adducts begins with liquid-phase extraction to separate proteins from lipid membranes, allowing measurement of both IsoK/LG protein adducts and F2-IsoP from the same sample if desired. IsoK/LG-lysyl-lactam adducts are measured by liquid chromatography tandem mass spectrometry after proteolytic digestion of extracted proteins, solid-phase extraction and preparative HPLC.

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Figure 1
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Figure 5: Reaction pathways utilized in the preparation of IsoK/LG by various methods.
Figure 6: Representative LC/MS/MS SRM chromatographs.

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References

  1. Roberts, L.J. II, Fessel, J.P., & Davies, S.S. The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation. Brain Pathol. 15, 143–148 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Davies, S.S., Amarnath, V. & Roberts, L.J. II Isoketals highly reactive gamma-ketoaldehydes formed from the H2-isoprostane pathway. Chem. Phys. Lipids 128, 85–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Salomon, R.G., Miller, D.B., Zagorski, M.G. & Coughlin, D.J. Solvent induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution. J. Am. Chem. Soc. 106, 6049–6060 (1984).

    Article  CAS  Google Scholar 

  4. Boutaud, O. et al. Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation. J. Biol. Chem. 278, 16926–16928 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Salomon, R.G. & Miller, D.B. Levuglandins: isolation, characterization, and total synthesis of new secoprostanoid products from prostaglandin endoperoxides. Adv. Prostaglandin Thromboxane Leukot. Res. 15, 323–326 (1985).

    CAS  PubMed  Google Scholar 

  6. Milne, G.L., Sanchez, S.C., Musiek, E.S. & Morrow, J.D. Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat. Protoc. 2, 221–226 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Morrow, J.D., Awad, J.A., Boss, H.J., Blair, I.A. & Roberts, L.J. II Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc. Natl. Acad. Sci. USA 89, 10721–10725 (1992).

    Article  CAS  PubMed  Google Scholar 

  8. Brame, C.J. et al. Modification of proteins by isoketal-containing oxidized phospholipids. J. Biol. Chem. 279, 13447–13451 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Moore, K. Isoprostanes and the liver. Chem. Phys. Lipids 128, 125–133 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Morrow, J.D. et al. Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation. J. Clin. Invest. 90, 2502–2507 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Salomon, R.G., Jirousek, M.R., Ghosh, S. & Sharma, R.B. Prostaglandin endoperoxides 21. Covalent binding of levuglandin E2 with proteins. Prostaglandins 34, 643–656 (1987).

    Article  CAS  PubMed  Google Scholar 

  12. Brame, C.J., Salomon, R.G., Morrow, J.D. & Roberts, L.J. II . Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts. J. Biol. Chem. 274, 13139–13146 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Iyer, R.S., Ghosh, S. & Salomon, R.G. Levuglandin E2 crosslinks proteins. Prostaglandins 37, 471–480 (1989).

    Article  CAS  PubMed  Google Scholar 

  14. Salomon, R.G. et al. Isolevuglandin–protein adducts in humans: products of free radical-induced lipid oxidation through the isoprostane pathway. Biochim. Biophys. Acta 1485, 225–235 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Fukuda, K. et al. Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels. Circ. Res. 97, 1262–1269 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Zagol-Ikapitte, I. et al. Prostaglandin H(2)-derived adducts of proteins correlate with Alzheimer's disease severity. J. Neurochem. 94, 1140–1145 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Davies, S.S. et al. Localization of isoketal adducts in vivo using a single-chain antibody. Free Radic. Biol. Med. 36, 1163–1174 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Talati, M. et al. Oxidant stress modulates murine allergic airway responses. Free Radic. Biol. Med. 40, 1210–1219 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Poliakov, E. et al. Isolevuglandins a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo. FASEB J. 17, 2209–2220 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Murthi, K.K., Salomon, R.G. & Sternlicht, H. Levuglandin E2 inhibits mitosis and microtubule assembly. Prostaglandins 39, 611–622 (1990).

    Article  CAS  PubMed  Google Scholar 

  21. Schmidley, J.W., Dadson, J., Iyer, R.S. & Salomon, R.G. Brain tissue injury and blood-brain barrier opening induced by injection of LGE2 or PGE2. Prostaglandins Leukot. Essent. Fatty Acids 47, 105–110 (1992).

    Article  CAS  PubMed  Google Scholar 

  22. Hoppe, G., Subbanagounder, G., O'Neil, J., Salomon, R.G. & Hoff, H.F. Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid. Biochim. Biophys. Acta 1344, 1–5 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Davies, S.S. et al. Effects of reactive gamma-ketoaldehydes formed by the isoprostane pathway (isoketals) and cyclooxygenase pathway (levuglandins) on proteasome function. FASEB J. 16, 715–717 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Boutaud, O., Montine, T.J., Chang, L., Klein, W.L. & Oates, J.A. PGH2-derived levuglandin adducts increase the neurotoxicity of amyloid beta1-42. J. Neurochem. 96, 917–923 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Amarnath, V., Amarnath, K., Amarnath, K., Davies, S. & Roberts, L.J. II . Pyridoxamine an extremely potent scavenger of 1,4-dicarbonyls. Chem. Res. Toxicol. 17, 410–415 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Davies, S.S. et al. Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H(2)O(2)-mediated cytotoxicity. Biochemistry 45, 15756–15767 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Amarnath, V., Amarnath, K., Matherson, T., Davies, S. & Roberts, L.J. A simplified synthesis of diastereomers of levuglandin E2. Synthetic Commun. 35, 397–408 (2005).

    Article  CAS  Google Scholar 

  28. Boutaud, O., Aronoff, D.M., Richardson, J.H., Marnett, L.J. & Oates, J.A. Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H2 synthases 99, 7130–7135 (2002).

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Authors and Affiliations

  1. Department of Pharmacology, Vanderbilt University, Nashville, 27232-6602, Tennessee, USA

    Sean S Davies & Olivier Boutaud

  2. Department of Pathology, Vanderbilt University, Nashville, 27232-6602, Tennessee, USA

    Venkataraman Amarnath

  3. Department of Biology, Centenary College, Shreveport, Los Angeles, 71104, California, USA

    Cynthia J Brame

  4. Department of Pharmacology and Medicine, Vanderbilt University, Nashville, 27232-6602, Tennessee, USA

    L Jackson Roberts II

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Correspondence to L Jackson Roberts II.

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Davies, S., Amarnath, V., Brame, C. et al. Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin γ-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry. Nat Protoc 2, 2079–2091 (2007). https://doi.org/10.1038/nprot.2007.298

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