Electrospray ionization and tandem mass spectrometry of cysteinyl eicosanoids: leukotriene C4 and FOG7.

The cysteinyl leukotrienes, LTC4, LTD4 and LTE4, and the recently described cysteinyl eicosanoid, 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) have been analyzed by tandem mass spectrometry. Both [M-H]- and [M+H]+ ions were produced by electrospray ionization and collision-induced dissociation of these molecular ion species were studied using both an ion trap and a triple quadrupole instrument. Product ion spectra obtained were characteristic of the structure of the cysteinyl leukotrienes and mechanisms of ion formation were investigated by using deuterium-labeled analogs. The product ion spectrum obtained following collision-induced dissociation of the [M-H]- anion from FOG7 was devoid of significant structural information and further studies of collision activation of the [M+H]+ spectrum were therefore examined. Positive ion MS3 spectra obtained in the ion trap from the gamma-glutamate cleavage products of FOG7 and its derivative (d7-FOG7) afforded an abundant ion not observed in spectra generated from the cysteinyl leukotrienes. Formation of this fragment ion likely occurred via a McLafferty-type rearrangement to afford cleavage of the C6-C7 bond adjacent to the sulfur atom and was valuable for the identification of the structure of FOG7 and defining the biosynthetic pathway as a 1,4-Michael addition of glutathione to 5-oxo-eicosatetraenoic acid (5-oxo-ETE).

Synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid and identification of novel omega-oxidized metabolites in the mouse macrophage.

The metabolism of arachidonic acid by the 5-lipoxygenase pathway not only leads to the formation of leukotrienes but also to the biologically active eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The synthesis of 5-oxo-ETE was investigated in the elicited peritoneal macrophage and the formation of 5-hydroxyeicosatetraenoic acid (5-HETE) as well as 5-oxo-ETE was quantitated using stable isotope dilution tandem mass spectrometry. The metabolism of 5-oxo-ETE in these same cells led to the formation of a series of novel less lipophilic metabolites oxidized near the methyl terminus that were structurally characterized using electrospray LC/MS and LC/MS/MS. Five novel metabolites of 5-oxo-ETE were identified including 5,18-diHETE, 5,19-diHETE, 5-oxo-19-HETrE, 5-oxo-18-HETrE, and 5,19-diHETrE. These metabolites corresponded to omega-1 and omega-2 oxidation of 5-oxo-ETE presumably formed by a specific cytochrome P450. There was no evidence for the formation of omega-oxidation (20-hydroxy metabolites), which are known products of metabolism of 5-oxo-ETE in other cell types. None of the metabolites were found to elevate intracellular calcium release, suggesting that this metabolic pathway may result in inactivation of 5-oxo-ETE. This is the first report of the biosynthesis of 5-oxo-ETE by tissue resident cell outside of the blood and the formation of novel omega-1 and omega-2 oxidation of this eicosanoid.

A novel glutathione containing eicosanoid (FOG7) chemotactic for human granulocytes.

A biologically active glutathione adduct of the eicosanoid 5-oxo-eicosatetraenoic acid has been observed as a product formed within the murine peritoneal macrophage. This five-oxo glutathione adduct (FOG(7)) was structurally characterized using electrospray tandem mass spectrometry as a 1,4 Michael addition product 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid. FOG(7) was found to be highly potent in stimulating eosinophil as well as neutrophil chemotaxis, also capable of initiating actin polymerization, without elevating intracellular free calcium ion concentration within either the eosinophil or polymorphonuclear leukocyte. These biological responses suggest that either FOG(7) activates a subset of receptors mediating the broader biological activity of the parent eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) or that a receptor not activated by 5-oxo-ETE participates in the chemotactic activity of FOG(7). The only other known biologically active glutathione adduct has been leukotriene C(4) (LTC(4)), another eicosanoid that exerts potent effects through the Cys-LT receptor. The biochemical parallel between the formation of LTC(4) and FOG(7) suggests an interesting mechanism by which biologically active eicosanoids derived from electrophilic intermediates may have unique distribution and prolonged efficacy in vivo.