Separation and Characterization of Cholesteryl Oxo- and Hydroxy-Linoleate Isolated from Human Atherosclerotic Plaque

In previous work we demonstrated that up to 30% of cholesteryl linoleate in homogenates of advanced human plaque samples is present in oxidized 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 cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. Enzymatic hydrolysis and measurement of liberated cholesterol and disappearance of the esters revealed that almost all of the material consisted of unoxidized cholesterol esterified to oxidized derivatives of octadecadienoate. 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 isolated compounds was identified, by co-chromatography with authentic standards, UV spectroscopy and chemical ionization and electron impact mass spectrometry, as cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. These oxidized fatty acids have been observed previously in plaque, in agreement with our new unambiguous demonstration of their presence as cholesteryl esters. The application of the methods described for the separation of the various forms of oxidized cholesteryl octadecadienoate may aid mechanistic studies of in vitro and in vivo lipoprotein lipid oxidation.