Oxidative inactivation of leukotriene C 4 by stimulated human polymorphonuclear leukocytes

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Abstract
Leukotriene C4 (LTC4) was metabolized by human polymorphonuclear leukocytes (PMNs) stimulated with phorbol myristate acetate (PMA) into three sets of products. These products differed in mobility on reverse-phase high-performance liquid chromatography (RP HPLC) from LTC4 and also from leukotriene D4 (LTD4) and leukotriene E4 (LTE4), the sequential products of peptide cleavage of LTC4. Products I, II, and III were eluted as doublets with an average retention time for each doublet of 7.5 ± 0.3, 10.5 ± 0.6, and 16.3 ± 1.1 min (mean ± SD), respectively, as compared with 13.8 min for LTC4. Doublet I material was biologically inactive and showed 4, doublet II material had 1% of the spasmogenic activity of LTC4 on the guinea pig ileum and was equally immunoreactive, and doublet III material was neither biologically active nor immunoreactive. When [14,15-3H]LTC4 and [35S]LTC4 were metabolized, all three doublet products retained the 3H label, whereas only the doublet I and doublet II products retained the 35S label. The UV absorbance spectra of the three sets of metabolites were as follows: doublet I, maximum at 280 nm with shoulders at about 270 and 290 nm; doublet II, maximum at 284.5 nm with shoulders at about 275 and 295 nm; and doublet III, maximum at 269 nm with shoulders at about 259 and 279 nm. The metabolism of LTC4 to the three classes of functionally inactive products by stimulated PMNs was completely blocked by catalase and azide, indicating a requirement for H2O2 and myeloperoxidase. When hypochlorous acid (HOCl)—considered to be a natural product of the interaction of myeloperoxidase, H2O2, and chloride ion—was formed chemically and allowed to react with LTC4, the resulting products were indistinguishable by UV and HPLC analyses from the doublet II and doublet III metabolites of LTC4. The doublet II products were identified as the two diastereoisomeric sulfoxides of LTC4 by comparison with synthetic reference compounds. The doublet III products were shown to be identical with synthetic samples of (5S, 12S)- and (5S, 12R)-6-trans-LTB4. The formation of two diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 can be explained in terms of an S-chlorosulfonium ion as the initial reactive intermediate, which subsequently undergoes conversion to product II by hydrolysis and product III by carbocation formation.