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Quantitative Analysis of Eicosanoids and Other Oxylipins

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A Practical Guide to Metabolomics Applications in Health and Disease

Part of the book series: Learning Materials in Biosciences ((LMB))

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Abstract

Eicosanoids and other oxylipins are oxygenation products from polyunsaturated fatty acids (PUFA). They can be formed non-enzymatically by (lipid) autoxidation as well as by specific enzymatic reactions. In mammals, enzymatic formation comprises three main pathways catalyzed by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases (CYP). Several of the oxylipins are highly potent lipid mediators, playing a key role in the regulation of pain, fever, and inflammation. Thus, oxylipin formation is a major drug target. In fact, common pharmaceuticals being sold without a prescription, e.g., non-steroidal anti-inflammatory drugs (NSAID), directly target COX.

In this chapter, a detailed protocol is described to investigate oxylipin formation during autoxidation and upon an inflammatory stimulus in cell culture. The low-abundant lipid mediators are quantified by means of targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experiments demonstrate that oxylipins are markers of cell biology, reflecting the redox status/oxidative stress on the one hand and the cellular inflammatory response on the other.

Based on a selected set of oxylipins the students learn how to set up a quantitative LC-MS/MS method and apply it to biological samples. With the detailed protocol, cell culture experiments can be rapidly set up in the laboratory to investigate the effects of oxidative stress or inflammatory stimuli in cells. The strategy also allows to test new compounds for their efficacy to reduce oxidative stress or to alleviate proinflammatory lipid mediator formation. Thus, the described procedures facilitate the implementation of individual research projects in advanced practical student courses.

Overall, the experiments lead to a comprehensive understanding of quantitative LC-MS/MS as well as basic cell culture experiments to study the bioactivity of new compounds. The investigation and interpretation of the results enable to understand oxylipin formation and their role in biology.

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Abbreviations

4-HNE:

4-Hydroxy-2-nonenal

ARA:

Arachidonic acid (C20:4 n6)

BCA:

Bicinchoninic acid

BHT:

Butylated hydroxytoluene

CE:

Collision energy

COX:

Cyclooxygenase

CXP:

Collision cell exit potential

CYP:

Cytochrome P450 monooxygenase

DHA:

Docosahexaenoic acid (C22:6 n3)

DiHETE:

Dihydroxy-eicosatetraenoic acid

DiHETrE:

Dihydroxy-eicosatrienoic acid

DMEM:

Dulbecco’s modified Eagle medium

DMSO:

Dimethyl sulfoxide

DP:

Declustering potential

EDTA:

Ethylenediaminetetraacetic acid

EP:

Entrance potential

EPA:

Eicosapentaenoic acid (C20:5 n3)

EpETrE:

Epoxy-eicosatrienoic acid

ESI:

Electrospray ionization

EtOH:

Ethanol

FCS:

Fetal calf serum

GIT:

Gastrointestinal tract

HAc:

Acetic acid

HETE:

Hydroxy-eicosatetraenoic acid

HHT/HHTrE:

Hydroxy-heptadecatrienoic acid

HpETE:

Hydroperoxy-eicosatetraenoic acid; hydroperoxy-ARA

IC50:

Half maximal inhibitory concentration

IS:

Internal standard

IsoP:

Isoprostane

KOH:

Potassium hydroxide

LC:

Liquid chromatography

LLOQ:

Lower limit of quantification

LOD:

Limit of detection

LOX:

Lipoxygenase

LPS:

Lipopolysaccharide

LT:

Leukotriene

LTA4:

5S-Trans-5,6-oxido-7E,9E,11Z,14Z-eicosatetraenoic acid

LTAH:

Leukotriene A4 hydrolase

LTB4:

5S,12R-Dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid

m/z:

Mass-to-charge ratio

MDA:

Malondialdehyde

MeOH:

Methanol

MRM:

Multiple reaction monitoring

MS :

Mass spectrometry

MS/MS:

Tandem mass spectrometry

n3-PUFA:

Omega-3 polyunsaturated fatty acid

n6-PUFA:

Omega-6 polyunsaturated fatty acid

NSAID:

Non-steroidal anti-inflammatory drug

PBS:

Phosphate buffered saline

PCR:

Polymerase chain reaction

PG:

Prostaglandin

PGF2α:

9α,11α,15S-Trihydroxy-prosta-5Z,13E-dien-1-oic acid

PUFA:

Polyunsaturated fatty acid

ROOH:

Organic hydroperoxide

RP:

Reversed-phase

rpm:

Rounds per minute

RPMI:

Roswell Park Memorial Institute (cell culture medium)

RSD:

Relative standard deviation

S/N:

Signal-to-noise ratio

SD:

Standard deviation

SPE:

Solid phase extraction

SRM:

Selected reaction monitoring

t-AUCB:

Trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid

t-BOOH:

Tert-butyl hydroperoxide

TGF-β1:

Transforming growth factor-β1

tR:

Retention time

TX:

Thromboxane

ULOQ:

Upper limit of quantification

UPLC:

Ultra-high-performance liquid chromatography

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Authors and Affiliations

  1. Chair of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Wuppertal, Germany

    Katharina M. Rund & Nils Helge Schebb

Authors
  1. Katharina M. Rund
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  2. Nils Helge Schebb
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Correspondence to Nils Helge Schebb .

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Editors and Affiliations

  1. Faculty of Biology & Medicine, University of Lausanne, Lausanne, Switzerland

    Julijana Ivanisevic

  2. Center for Proteomics & Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

    Martin Giera

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Rund, K.M., Schebb, N.H. (2023). Quantitative Analysis of Eicosanoids and Other Oxylipins. In: Ivanisevic, J., Giera, M. (eds) A Practical Guide to Metabolomics Applications in Health and Disease. Learning Materials in Biosciences. Springer, Cham. https://doi.org/10.1007/978-3-031-44256-8_13

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  • DOI: https://doi.org/10.1007/978-3-031-44256-8_13

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