HYPOCHLOROUS ACID-MEDIATED MODIFICATION OF CHOLESTEROL AND PHOSPHOLIPID - ANALYSIS OF REACTION-PRODUCTS BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

Oxidative modification of membrane lipids by hypochlorous acid could b e an important clement in the mechanism of membrane disruption by acti vated neutrophils. We have previously shown that hypochlorous acid rea cts with unsaturated fatty acids of membrane phospholipids to give fat ty acid chlorohydrins (Winterbourn et al. 1992. Arch. Biochem. Biophys . 296: 547-555). In the present study, we examined the reaction of cho lesterol in bilayers with an inert phospholipid carrier. Product separ ation and identification was performed using gas chromatography-mass s pectrometry after trimethylsilyl-derivatization. Unlike the reaction o f hypochlorous acid with unsaturated fatty acids, no chlorohydrin deri vatives were found with cholesterol. Instead, the main oxidation produ cts were identified as the epimeric cholesterol 5,6-epoxides and 4-hyd roxycholesterol, while several other hydroxy- and keto-derivatives wer e also found in smaller amounts. Analysis of the products obtained aft er reaction of vesicles composed of a mixture of several unsaturated p hospholipid species plus cholesterol revealed that the individual fatt y acids and cholesterol all exhibit comparable susceptibilities toward hypochlorous acid. Using myeloperoxidase to generate hypochlorous aci d, basically the same products and product distribution were obtained. These studies show that unsaturated phospholipids and cholesterol can be profoundly modified by reaction with hypochlorous acid. This warra nts further investigation to define the role of lipid modifications in neutrophil-mediated membrane disruption.