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Quantitative assays for esterified oxylipins generated by immune cells

Nature Protocols volume 5, pages 1919–1931 (2010)Cite this article

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Abstract

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins—in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids—attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC–tandem mass spectrometry (LC/MS/MS) in multiple reaction–monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ∼2 d. Following this, extraction of 30 samples takes ∼3 h, with LC/MS/MS run time of 50 min per sample.

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Figure 1
Figure 2
Figure 3: Purification of 18:0a/15-HpETE-PE using reverse-phase HPLC-UV.
Figure 4: Purification of 18:0a/15-HETE-PE using reverse-phase HPLC-UV.
Figure 5: LC/MS/MS of a mixture of standards comprising six positional isomers of 16:0a/HETE-PC and 18:0a/HETE-PE.
Figure 6: LC/MS/MS of a mixture of standards comprising 10 positional isomers of 18:0a/HDOHE-PE.
Figure 7: LC/MS/MS of a mixture of standards comprising two positional isomers of 16:0a/HODE-PE.
Figure 8: LC/MS/MS and standard curve for 18:0a/15-KETE-PE.
Figure 9: Generation of standard curves for 5-, 12- and 15-HETE-PE isomers.
Figure 10: Detection of HETE-PEs and PCs in activated immune cells using LC/MS/MS.

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Author notes

  1. Alwena H Morgan and Victoria J Hammond: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK

    Alwena H Morgan, Victoria J Hammond, Lloyd Morgan, Christopher P Thomas, Yoel R Garcia-Diaz & Valerie B O'Donnell

  2. Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    Keri A Tallman & Ned A Porter

  3. School of Pharmacy, Cardiff University, Cardiff, UK

    Christopher McGuigan & Michaela Serpi

  4. Department of Pharmacology, University of Colorado at Denver, Aurora, Colorado, USA

    Robert C Murphy

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  1. Alwena H Morgan
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Contributions

V.B.O. designed experiments and wrote the article. V.J.H., A.H.M., L.M. and C.P.T. conducted experiments. C.M. and M.S. conducted NMR analysis. K.A.T., Y.R.G.D. and N.A.P. contributed to standard synthesis. R.C.M. advised on LC/MS/MS methodologies.

Corresponding author

Correspondence to Valerie B O'Donnell.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Double bands region of the 13C-NMR spectra (125 MHz) of purified 15-HETE- recorded on a Bruker Avance 500 MHz instrument at 25 °C. (PDF 139 kb)

Supplementary Figure 2

31P-NMR spectra (202 MHz) of purified 15-HETE-PE recorded on a Bruker Avance 500 MHz instrument at 25 °C (PDF 139 kb)

Supplementary Figure 3

1H-NMR spectra (500 MHz) of purified 15-HETE-PE recorded on a Bruker Avance 500 MHz instrument at 25 °C (PDF 196 kb)

Supplementary Figure 4

MS/MS spectra of 12- and 15-HETE-PLs. (PDF 24 kb)

Supplementary Figure 5

MS/MS spectrum of 15-HpETE-PE and15-KETE-PE. (PDF 22 kb)

Supplementary Table 1

Structural data on standards. (DOC 30 kb)

Supplementary Table 2

Assay parameters for standards. (DOC 67 kb)

Supplementary Method

Method for obtaining structural data on standards (DOC 57 kb)

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Morgan, A., Hammond, V., Morgan, L. et al. Quantitative assays for esterified oxylipins generated by immune cells. Nat Protoc 5, 1919–1931 (2010). https://doi.org/10.1038/nprot.2010.162

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