12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C synthase (LTCS). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTCS, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 μM), which confirms the involvement of pt12-LOX. Interestingly, TOG was formed independently of LTCS in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTCS. Nevertheless, TOG can also be formed by LTCS as the biosynthesis in HEK_12-LOX_LTCS cells was reduced by the specific LTCS inhibitor TK04a. In summary, TOG was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.