Skip to main content

Advertisement

SpringerLink
Log in

A sensitive LC-MS/MS method for the quantification of regioisomers of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in human plasma during endothelial stimulation

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid–liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Bellien J, Joannides R, Richard V, Thuillez C. Modulation of cytochrome-derived epoxyeicosatrienoic acids pathway: a promising pharmacological approach to prevent endothelial dysfunction in cardiovascular diseases? Pharmacol Ther. 2011;131:1–17.

    Article  CAS  Google Scholar 

  2. Duflot T, Roche C, Lamoureux F, Guerrot D, Bellien J. Design and discovery of soluble epoxide hydrolase inhibitors for the treatment of cardiovascular diseases. Expert Opin Drug Discov. 2014;9:229–43.

    Article  CAS  Google Scholar 

  3. Morisseau C, Hammock BD. Impact of soluble epoxide hydrolase and epoxyeicosanoids on human health. Annu Rev Pharmacol Toxicol. 2013;53:37–58.

    Article  CAS  Google Scholar 

  4. Bellien J, Iacob M, Remy-Jouet I, Lucas D, Monteil C, Gutierrez L, et al. Epoxyeicosatrienoic acids contribute with altered NO and endothelin-1 pathways to conduit artery endothelial dysfunction in essential hypertension. Circulation. 2012;125:1266–75.

    Article  CAS  Google Scholar 

  5. Chen D, Whitcomb R, MacIntyre E, Tran V, Do ZN, Sabry J, et al. Pharmacokinetics and pharmacodynamics of AR9281, an inhibitor of soluble epoxide hydrolase, in single- and multiple-dose studies in healthy human subjects. J Clin Pharmacol. 2012;52:319–28.

    Article  CAS  Google Scholar 

  6. Lazaar AL, Yang L, Boardley RL, Goyal NS, Robertson J, Baldwin SJ, et al. Pharmacokinetics, pharmacodynamics and adverse event profile of GSK2256294, a novel soluble epoxide hydrolase inhibitor. Br J Clin Pharmacol. 2016;81:971–9.

    Article  CAS  Google Scholar 

  7. Karara A, Wei S, Spady D, Swift L, Capdevila JH, Falck JR. Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma. Biochem Biophys Res Commun. 1992;182:1320–5.

    Article  CAS  Google Scholar 

  8. Karara A, Dishman E, Falck JR, Capdevila JH. Endogenous epoxyeicosatrienoyl-phospholipids. J Biol Chem. 1991;266:7561–9.

    CAS  Google Scholar 

  9. Goulitquer S, Dréano Y, Berthou F, Corcos L, Lucas D. Determination of epoxyeicosatrienoic acids in human red blood cells and plasma by GC/MS in the NICI mode. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;876:83–8.

    Article  CAS  Google Scholar 

  10. Fang X, Weintraub NL, Spector AA. Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells. Prostaglandins Other Lipid Mediat. 2003;71:33–42.

    Article  CAS  Google Scholar 

  11. Schuchardt JP, Schmidt S, Kressel G, Dong H, Willenberg I, Hammock BD, et al. Comparison of free serum oxylipin concentrations in hyper- vs. normolipidemic men. Prostaglandins Leukot Essent Fatty Acids. 2013;89:19–29.

    Article  CAS  Google Scholar 

  12. Schebb NH, Ostermann AI, Yang J, Hammock BD, Hahn A, Schuchardt JP. Comparison of the effects of long-chain omega-3 fatty acid supplementation on plasma levels of free and esterified oxylipins. Prostaglandins Other Lipid Mediat. 2014;113-5:21–9.

    Article  Google Scholar 

  13. Tsikas D, Zoerner AA. Analysis of eicosanoids by LC-MS/MS and GC-MS/MS: a historical retrospect and a discussion. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;964:79–88.

    Article  CAS  Google Scholar 

  14. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–7.

    Article  CAS  Google Scholar 

  15. FDA Guidance: Guidance for industry. Bioanalytical method validation. 2001. http://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf. Accessed 30 Jun 2016.

  16. EMA Guideline: Guideline on bioanalytical method validation. 2011. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. Accessed 30 Jun 2016.

  17. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30.

    Article  CAS  Google Scholar 

  18. Joannides R, Costentin A, Iacob M, Compagnon P, Lahary A, Thuillez C. Influence of vascular dimension on gender difference in flow-dependent dilatation of peripheral conduit arteries. Am J Physiol Heart Circ Physiol. 2002;282:H1262–9.

    Article  CAS  Google Scholar 

  19. Fulton D, Falck JR, McGiff JC, Carroll MA, Quilley J. A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol. J Lipid Res. 1998;39:1713–21.

    CAS  Google Scholar 

  20. Carroll MA, Garcia MP, Falck JR, McGiff JC. 5,6-epoxyeicosatrienoic acid, a novel arachidonate metabolite. Mechanism of vasoactivity in the rat. Circ Res. 1990;67:1082–8.

    Article  CAS  Google Scholar 

  21. Van de Merbel NC. Quantitative determination of endogenous compounds in biological samples using chromatographic techniques. Trends Anal Chem. 2008;27:924–33.

    Article  Google Scholar 

  22. Willenberg I, Ostermann AI, Schebb NH. Targeted metabolomics of the arachidonic acid cascade: current state and challenges of LC-MS analysis of oxylipins. Anal Bioanal Chem. 2015;407:2675–83.

    Article  CAS  Google Scholar 

  23. Fang X, VanRollins M, Kaduce TL, Spector AA. Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells. J Lipid Res. 1995;36:1236–46.

    CAS  Google Scholar 

  24. Levison BS, Zhang R, Wang Z, Fu X, DiDonato JA, Hazen SL. Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry. Free Radic Biol Med. 2013;59:2–13.

    Article  CAS  Google Scholar 

  25. Zhu P, Peck B, Licea-Perez H, Callahan JF, Booth-Genthe C. Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activity in vivo. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879:2487–93.

    Article  CAS  Google Scholar 

  26. Capdevila JH, Falck JR, Harris RC. Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase. J Lipid Res. 2000;41:163–81.

    CAS  Google Scholar 

  27. Archer SL, Gragasin FS, Wu X, Wang S, McMurtry S, Kim DH, et al. Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels. Circulation. 2003;107:769–76.

    Article  CAS  Google Scholar 

  28. Zhao X, Pollock DM, Inscho EW, Zeldin DC, Imig JD. Decreased renal cytochrome P450 2C enzymes and impaired vasodilation are associated with angiotensin salt-sensitive hypertension. Hypertension. 2003;41:709–14.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the technicians from the Unit of Pharmacokinetics and Toxicology of the Rouen University Hospital for their assistance. This work was funded by a grant from the Fondation de France (eng 2011-00020459).

Author information

Authors and Affiliations

  1. Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France

    Thomas Duflot, Tony Pereira, Michèle Iacob, Christian Thuillez, Patricia Compagnon, Robinson Joannidès, Fabien Lamoureux & Jérémy Bellien

  2. Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France

    Thomas Duflot, Clothilde Roche, Christian Thuillez, Robinson Joannidès, Fabien Lamoureux & Jérémy Bellien

  3. Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France

    Thomas Duflot, Clothilde Roche, Christian Thuillez, Robinson Joannidès, Fabien Lamoureux & Jérémy Bellien

  4. Normandie Université, Laboratoire SMS – EA3233, Université de Rouen, 76281, Mont Saint Aignan, France

    Pascal Cardinael & Najla El-Gharbi Hamza

Authors
  1. Thomas Duflot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tony Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Clothilde Roche
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michèle Iacob
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pascal Cardinael
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Najla El-Gharbi Hamza
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christian Thuillez
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Patricia Compagnon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robinson Joannidès
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Fabien Lamoureux
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jérémy Bellien
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jérémy Bellien.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 272 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Duflot, T., Pereira, T., Roche, C. et al. A sensitive LC-MS/MS method for the quantification of regioisomers of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in human plasma during endothelial stimulation. Anal Bioanal Chem 409, 1845–1855 (2017). https://doi.org/10.1007/s00216-016-0129-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-0129-1

Keywords

Search

Navigation