SEPARATION AND CHARACTERIZATION OF CHOLESTERYL OXO-LINOLEATE AND HYDROXY-LINOLEATE ISOLATED FROM HUMAN ATHEROSCLEROTIC PLAQUE

In previous work we demonstrated that up to 30 % of cholesteryl Linole ate in homogenates of advanced human plaque samples is present in oxid ized forms. Here we show that the material from plaque hexane extracts which co-elutes with cholesteryl hydroxy linoleate on reversed phase HPLC (Anal Biochem 1993;213:79), is composed of several isomers of cho lesteryl hydroxy- and cholesteryl oxo-octadecadienoate. Enzymatic hydr olysis and measurement of liberated cholesterol and disappearance of t he esters revealed that almost all of the material consisted of unoxid ized cholesterol esterified to oxidized derivatives of octadecadienoat e. Semi-preparative reversed-phase HPLC was used to obtain sufficient quantities of this co-eluting material to undertake normal phase HPLC separation of these components. The nature of such separated and isola ted compounds was identified, by co-chromatography with authentic stan dards, UV spectroscopy and chemical ionization and electron impact mas s spectrometry, as cholesteryl hydroxy- and cholesteryl oxo-octadecadi enoate. These oxidized fatty acids have been observed previously in pl aque, in agreement with our new unambiguous demonstration of their pre sence as cholesteryl esters. The application of the methods described for the separation of the various forms of oxidized cholesteryl octade cadienoate may aid mechanistic studies of in vitro and in vivo lipopro tein lipid oxidation.