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Merck
CN

Quantitative assays for esterified oxylipins generated by immune cells.

Nature protocols (2010-12-04)
Alwena H Morgan, Victoria J Hammond, Lloyd Morgan, Christopher P Thomas, Keri A Tallman, Yoel R Garcia-Diaz, Christopher McGuigan, Michaela Serpi, Ned A Porter, Robert C Murphy, Valerie B O'Donnell
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.

MATERIALS
Product Number
Brand
Product Description

Avanti
18:0-20:4 PE, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine, chloroform
Sigma-Aldrich
Dichloromethane, anhydrous, ≥99.8%, contains 40-150 ppm amylene as stabilizer
Avanti
18:0-22:6 PE, 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, chloroform
Avanti
16:0-18:2 PE, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, chloroform
Avanti
16:0-20:4 PC, Avanti Research - A Croda Brand
Avanti
14:0 PE, Avanti Research - A Croda Brand
Avanti
14:0 PC (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine, chloroform
Avanti
14:0 PC (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine, powder