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Mass spectrometry characterization of thiol conjugates linked to poly-oxygenated PUFA species.

Radical mediated oxidation of polyunsaturated fatty acids (PUFA) are known to generate a series of poly-oxygenated cyclic products (PUFA-On, n≥3). Here we describe the characterization of glutathione (GSH)-conjugates bound to poly-oxygenated docosahexaenoic (DHA-On, n=3-9), arachidonic (ARA-On, n=3-7), α-linolenic (ALA-O3) and linoleic (LA-O3) acid species. Similar conjugates were also characterized for N-acetylcysteine (NAC) and Cu,Zn-superoxide dismutase (SOD1). Extensive LC-MS/MS characterization using a synthetic α-linolenic hydroxy-endoperoxide (ALA-O3) derivative revealed at least two types of mechanisms leading to thiol adduction. A mechanism involving the nucleophilic attack by thiolate anion on 1,2-dioxolane to form a sulfenate ester-bonded conjugate and a mechanism involving cleavage of the dioxolane to form a α,β-unsaturated carbonyl followed by Michael addition reaction. Finally, we detected a GSH conjugate with hydroxy-endoperoxide derived from linoleic acid (LA-O3) in mice liver. In summary, our study reveals the formation of series of thiol conjugates that are bound to highly oxygenated PUFA species. GSH conjugates described in our study may potentially play relevant roles in redox and inflammatory processes, especially under high oxygen tension conditions.