Identification of Glutathione Adducts of the Myeloperoxidase Oxidation Product of Plasmalogen, α‐Chlorofatty Aldehyde

Abstract

α‐Chlorofatty aldehydes (α‐ClFALD) are produced by hypochlorous acid attack on plasmalogens during neutrophil activation. In this study the reactivity of the α‐chlorinated carbon of α‐ClFALD with the nucleophile, glutathione (GSH) was characterized, and the product of this reaction was examined in activated human neutrophils. Utilizing electrospray ionization mass spectroscopy, the reaction product of GSH and the α‐ClFALD, 2‐chlorohexadecanal, was characterized. The resulting conjugate, hexadecanal‐GSH retains an intact free aldehyde while the α‐chlorinated carbon is covalently bound to the sulfhydryl of GSH resulting in chlorine ejection from this adduct. Stable isotope labeled [d4]‐ hexadecanal‐GSH was synthesized. [d4]‐hexadecanal‐GSH was used to further confirm the structure, and was used to quantify natural α‐ClFALD conjugates of GSH using reversed phase liquid chromatography with detection by ESI‐MS and selective reaction monitoring. The 2‐chlorohexadecanal conjugate of GSH, hexadecanal‐GSH, is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2‐chlorohexadecanal. Furthermore, phorbol myristate acetate (PMA) treated primary human neutrophils have elevated levels of hexadecanal‐GSH, octadecanal‐GSH, 2‐chlorohexadecanal and 2‐chlorooctadecanal levels, which are reduced in PMA treated neutrophils pretreated with aminotriazole. Together, these studies demonstrate for the first time that chlorinated fatty aldehydes are covalently modified by glutathione forming novel peptidoaldehydes, which may play an important role in inflammatory signaling.