CHLORINATION OF CHOLESTEROL IN CELL-MEMBRANES BY HYPOCHLOROUS ACID

Neutrophils and monocytes produce the highly cytotoxic hypochlorous ac id (HOCl) via the myeloperoxidase (MPO)-catalyzed reaction of H2O2 Wit h Cl- We have investigated the reaction of reagent and MPO-derived HOC l with cholesterol in a purified liposome system, as well as progressi vely more complex biological systems, The products were identified by thin-layer chromatography (TLC) and characterized by mass spectrometry (MS), TLC of the HOCl-treated samples gave four major cholesterol pro ducts with color development typical of oxysterols, Two of the product s coeluted with authentic alpha- and beta-chlorohydrin standards, As w as the case with the standards, they were readily converted into their respective epoxides during analysis by MS, Gas chromatography-mass sp ectrometry analysis of the other major product (band 3) gave peaks con sistent with epoxides as well as a late-eluting peak with a distinct m ass spectrum, Electrospray MS of this product confirmed its identity a s a chlorohydrin based on the presence of the predicted molecular ion and 3:1 Cl isotope ratios, Lipids extracted from red cells and isolate d red cell membranes were exposed to HOCl and gave identical products to the purified cholesterol liposome system as determined by TLC and M S, Higher concentrations of HOCl were required due to competition by o ther unsaturated lipids and protein molecules. When intact red cells, neutrophils, and MCF7 mammary carcinoma cells were exposed to HOCl, ch olesterol chlorohydrins were formed, as detected by TLC, The formation of cholesterol chlorohydrins could be potentially disruptive to cell membranes and result in cell lysis and death, They could also be poten tial biomarkers for oxidative damage associated with neutrophil/monocy te activation. (C) 1996 Academic Press, Inc.