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Although the pseudohalide thiocyanate (SCN−) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN−/H2O2 system and activated eosinophils as roughly equimolar amounts of OSCN−(hypothiocyanite) and OCN− (cyanate). Red blood cells exposed to increasing concentrations of OSCN−/OCN− are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN−/OCN− inactivates red blood cell membrane ATPases 10–1000 times more potently than do HOCl, HOBr, and H2O2. Exposure of glutathione S-transferase to [14C]OSCN−/OCN− causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN−/H2O2reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.

Isolevuglandins (isoLGs) are a family of reactive gamma-ketoaldehydes generated by free radical oxidation of arachidonate-containing lipids through the isoprostane pathway. Elevated plasma levels of isoLG protein adducts are observed in subjects with atherosclerosis compared with age/gender-matched controls. However, mechanisms for the generation of isoLGs in vivo are not established. Here we show that free radical-induced peroxidation promoted by the myeloperoxidase (MPO)/H2O2 system of leukocytes serves as one mechanism for the generation of isoLGs in vivo. Using a Candida sepsis model of inflammation, we demonstrate 3.5- and 2.7-fold increases in iso[4]LGE2 and isoLGE2 adducts of plasma proteins after pathogen exposure in wild-type mice. Plasma levels of F2 isoprostanes were not significantly increased after pathogen challenge in this model. MPO knockout mice demonstrated significant reductions (34%, P=0.003) in plasma levels of iso[4]LGE2 protein adducts after pathogen challenge compared with wild-type mice. Mass spectrometry and immunochemical methods demonstrate MPO-dependent formation of iso[4]LGE2 and isoLGE2 phospholipids and their corresponding isoLG protein adducts in model systems. The present studies thus identify MPO as one pathway for generation of isoLGs in vivo. They also suggest that long-lived protein isoLG adducts may serve as an alternative integrated sensor of oxidant stress in vivo.

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide ((.)NO(2)), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO(-/-)), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stress in vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F(2)-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F(2)-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO(-/-) mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO(-/-) group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and (.)NO(2), respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO(-/-) samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of (.)NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo.

A protein expressing phospholipase A2 activity was purified from the granular amebocyte of the horseshoe crab, Limulus polyphemus by cation-exchange, size-exclusion chromatography and semi-preparative reverse-phase–high pressure liquid chromatography (RP–HPLC). The protein had an apparent mass of 17.7 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but a more accurate estimate of 18.5 kDa was assigned by electrospray ionization–mass spectrometry (ESI–MS). A partial sequence of this protein demonstrated total sequence homology with an 18.5 kDa protein with cell aggregating properties from Limulus reported by Fujii et al. [J. Biol. Chem. 267:22452.]. In these studies, the Limulus protein demonstrated a positive cross-reaction to polyclonal anti-human recombinant phospholipase A2 (group II, 14 kDa). The protein did not display a significant loss of biological activity after boiling, but all enzymatic activity was lost after boiling in the presence of the reducing agent betamercaptoethanol (s-mercaptoethanol). The Limulus protein was inhibited by manoalide, a covalent irreversible phospholipase A2 inhibitor, in a dose-dependent fashion with 50% inhibition occurring at a concentration of 0.48 μM. The Limulus protein displayed no activity in a triglyceride lipase assay. These studies characterize an alternative phospholipase A2 activity for the previously described 18.5 kDa protein from the L. polyphemus amebocyte.