Hydroperoxide Formation of Steryl Ester

Phytosterols and -stanols are added to food products because of their known ability to lower serum cholesterol levels. They are applied either in their free or esterified forms, i.e. as fatty acid esters. Sterols are known to form variety of oxidation products under exposure to heat, light and metal contaminants, for example in food processing conditions. Since these oxides may have adverse health effects, the oxidation process needs to be studied. Until recently, sterol oxidation studies have concentrated on following the formation of secondary oxidation products in free sterol and steryl esters, but little is known about the oxidation of steryl esters as intact molecules. The aim of this experiment was to study primary autoxidation of intact steryl ester by measuring hydroperoxide formation in bulk cholesteryl ester. Cholesteryl linoleate was maintained at 60°C for 0-72 h after which formed hydroperoxides were determined with normal phase high performance liquid chromatography connected to diode array detector (HPLC-DAD). Also peroxide value (PV) was measured to indicate the total amount of formed hydroperoxides. With HPLC method steryl ester -OOH's could be analysed as intact esters, without saponification. A gradient elution was performed with 0.3-10% methyl-tert-butyl ether (MTBE) in heptane followed by cleanup with 30% MTBE. Compounds were detected with DAD at wavelengths 206 nm and 234 nm. Peroxide value indicated that the formation of hydroperoxides reached the maximum after 12 h of prolonged heating. According to HPLC data, at this time point less than 10% of the hydroperoxide groups were located in the sterol moiety and more than 90% in the fatty acid chain. The proportion of sterol-OOH's increased as the heating continued; at 24 h 20% and at 48 h 30%. However, after 72 h no hydroperoxides were observed. In conclusion, oxidation of cholesteryl linoleate started in the fatty acid moiety and as the reaction progressed more of the sterol -OOH's were observed, though at all time points fatty acid -OOH's were dominating.