Quantitative assays for esterified oxylipins generated by immune cells.
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.
Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK.
Alwena H
AH
Morgan
Victoria J
VJ
Hammond
Lloyd
L
Morgan
Christopher P
CP
Thomas
Keri A
KA
Tallman
Yoel R
YR
Garcia-Diaz
Christopher
C
McGuigan
Michaela
M
Serpi
Ned A
NA
Porter
Robert C
RC
Murphy
Valerie B
VB
O'Donnell
D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015394:Molecular Structure; D054883:Oxylipins / Q000302:isolation & purification*; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D054883:Oxylipins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine