Capillary gas chromatography for the assessment of cholesterol oxides in the heart

Recent studies indicated the presence of cholesterol oxides in biological tissues such as heart under pathophysiological conditions. Currently, no suitable method is available to separate and quantitate these oxides in biological tissues. This study was undertaken to develop a method suitable for the quantification of cholesterol oxides in heart. Since under normal conditions cholesterol oxidation does not occur, we exposed isolated perfused rat heart to oxygen-derived free radicals, which are known to oxidize cholesterol in heart. Free radical treated hearts were homogenized and lipids extracted by conventional techniques. The extracted lipids were saponified, and non-saponified portions were extracted with ether. Silyl derivatives of the extracted products were subjected to capillary gas chromatography. One microlitre of the sample was injected onto an SE 54 Supelco capillary column and run by stepwise temperature programming from 230 to 260 degrees C at a rate of 20 degrees C min-1, and then from 260 to 290 degrees C at 6 degrees C min-1. 5-Androsten-3 beta-ol-17-one was used as internal standard. The separated cholesterol oxides were identified by comparing with authentic standards. This method was suitable to separate 7 alpha-hydroxycholesterol, dihydroxycholesterol, 7 beta-hydroxycholesterol, 20 alpha-hydroxycholesterol, cholesterol-5 alpha,6 alpha-epoxide, and 5-cholestene in heart. Since some of these oxides are cytotoxic to the heart, identification of these oxides should be important to understand the pathophysiology of myocardial disease and for the successful therapy to prevent them.