DETERMINATION OF STEROLS, OXYSTEROLS, AND FATTY-ACIDS OF PHOSPHOLIPIDS IN CELLS AND LIPOPROTEINS - A ONE-SAMPLE METHOD

In addition to fatty acids, especially polyunsaturated species, choles terol oxidizes and leads to various oxygenated derivatives, named oxys terols. They display a wide range of adverse biological properties. Mo nitoring oxysterols is important in the evaluation of the potential ri sks associated with lipid oxidation. In the present study, a quick and reliable method was developed for analysis of oxysterols, sterols, an d fatty acid composition of phospholipids in the same biological sampl e. Total lipid extraction was determined after addition of several int ernal standards (epicoprostanol for sterols, 19-hydroxy-cholesterol fo r oxysterol and di-heptadecanoyl-phosphatidylcholine for phospholipid fatty acids). Cold acetone-mediated precipitation was then used to fra ctionate sterols from phospholipids. The phospholipid-containing preci pitate was transmethylated for fatty acid analysis by gas chromatograp hy. The sterol- and oxysterol-containing phase was saponified under mi ld conditions to avoid artificial oxysterol generation and was analyze d by gas chromatography after derivatization into trimethylsilyl ether s. The overall procedure was found to be specific with good recovery a nd reproducibility for sterols, oxysterols [mean coefficient of variat ion in percent (CV), 11.3%] as well as phospholipid fatty acids (CV, 5 .6%). This procedure has been used to document in vitro free radical t reated-human low-density lipoproteins and erythrocytes. Results demons trated that this method is a useful tool in assessing qualitative and quantitative differences in oxysterols and phospholipid fatty acid pat terns attributed to lipid oxidation.