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

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.

Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids

“…In line with a recent study (7), our chiral analysis showed that monohydroxy metabolites are generally present as racemates in the human circulation, whereas enantioselective responses occur after stimulating their de novo biosynthesis. Ca 2+ -ionophore stimulation resulted in specific increases of the (S)-enantiomers of those HETEs, HEPEs, and HDHAs that were apparently produced by the stereospecific actions of 5-, 12-, and 15-LOX enzymes expressed in different blood cells (48)(49)(50).…”

“…This method can be coupled with normal-phase chiral-LC and requires prior derivatization of oxylipins, e.g., into their pentafluorobenzyl esters (6). Using this sensitive approach, a recent study showed stereospecific responses to in vitro stimulation of human blood samples regarding the formation of HETEs (7). An alternative strategy takes advantage of chiral stationary phases that are compatible with water-containing polar mobile phases.…”

Mid-gestation serum lipidomic profile associations with spontaneous preterm birth are influenced by body mass index

“…leukotrienes) and triols (e.g. lipoxins) correlated strongly with the autooxidative markers (S6 Table) supporting an autooxidative origin [57,58]. A recent study of effects of long-term storage on the plasma metabolome reports general stability for up to 7yrs, with significant changes observed with additional time up to 16yrs [59].…”

Prominent release of lipoxygenase generated mediators in a murine house dust mite-induced asthma model

“…During inflammation, infiltrating leukocytes exert their actions in part by the release of oxidative species. Although isoprostanes are considered markers of free radical induced lipid peroxidation/oxidative reactions several of the PUFA-derived oxylipins can be formed by autoxidation, wherein equal amounts of the R and S-enantiomer is produced (Mazaleuskaya et al, 2018). However, both 5-LOX and 15-LOX enzymes stereo-selectively produce hydroxylated lipids with the S-configuration.…”

“…Chiral chromatography can therefore be essential to elucidate the biosynthetic source of production of specific lipid species. For example, 9(S)-and 9(R)-HETE were shown to be equally produced following lipopolysaccharide and/or zymosan stimulated human whole blood while 12(S)-and 15(S)-HETE were the main products (Mazaleuskaya et al, 2018).…”