cytellin and 7-ketocholesterol

7-Ketocholesterol and 7-ketositosterol were chosen as reliable markers of the oxidation of cholesterol and phytosterols in infant milk formulas and infant milk cereals. A reversed-phase HPLC method was developed to measure them simultaneously in infant formulas. This method was then tested on a wide range of infant milk formulas and milk cereals on sale in Italy whose lipid composition is representative of the most common commercial formulas. The analytical results revealed no significant differences in the extent of oxidation of cholesterol and sitosterol. As the level of 7-ketocholesterol often followed the cholesterol level, a cholesterol content similar to that of human milk produced amounts of cholesterol oxides with possible negative effects on infant health. In contrast, the low cholesterol content of milk cereals never produced amounts of cholesterol oxides high enough to cause concern. The contents of phytosterols and hence their oxides were always low.

Topics: Animals; Cholesterol; Chromatography, Gas; Chromatography, High Pressure Liquid; Edible Grain; Humans; Infant; Infant Food; Ketocholesterols; Lipids; Milk; Oxidation-Reduction; Phytosterols; Sitosterols; Sterols

7-Ketocholesterol (a major cholesterol oxidation product) and phytosterols are important indicators of lipoprotein oxidation and lipoprotein metabolism respectively. We describe a simple, sensitive and reproducible method for the simultaneous measurement of these sterols in human lipoprotein samples by capillary column gas liquid chromatography. The method is suitable for clinical studies as small quantities of lipoprotein are required. Sterols are analysed after extraction from lipoprotein samples obtained by sequential flotation ultracentrifugation. The method involves briefly: extraction from lipoprotein samples using chloroform-methanol, saponification of sterol esters using cold potassium hydroxide, purification and derivatisation to trimethylsilyl ethers using BSTFA and 1% TMCS. Oxidation is prevented by drying under nitrogen and the use of powerful antioxidants. Separation is achieved using a DB-1 capillary column and a two-stage temperature ramp from 180-250 degrees C and detection using FID. The identity of sterols can be confirmed by GC-MS. Phytosterols and 7-ketocholesterol are present at low concentration in all the major lipoproteins. Using [3,4-13C]cholesterol and GC-MS we present evidence that cholesterol oxidation does not occur during the processing of lipoproteins using this technique.

Topics: Capillary Action; Cholesterol; Chromatography, Gas; Humans; Ketocholesterols; Lipoproteins; Male; Oxidation-Reduction; Phytosterols; Reproducibility of Results; Sitosterols