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J Lipid Res. 2018 Mar;59(3):564-575. doi: 10.1194/jlr.D081414. Epub 2018 Jan 4.

Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS.

Author information

1
Institute for Translational Medicine and Therapeutics University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160.
2
Penn Superfund Research Program (SRP) Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160.
3
Institute for Translational Medicine and Therapeutics University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160 mesaros@pennmedicine.upenn.edu.

Abstract

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

KEYWORDS:

chiral hydroxyeicosatetraenoic acids; coagulation; human blood; hydroxyeicosatetraenoic acids; plasma lipidomics; serum lipidomics; ultra-high-performance liquid chromatography-electron capture atmospheric pressure chemical ionization high-resolution mass spectrometry

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