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, cholesterol oxidizes and leads to various oxygenated derivatives, named oxysterols. They display a wide range of adverse biological properties. Monitoring oxysterols is important in the evaluation of the potential risks associated with lipid oxidation. In the present study, a quick and reliable method was developed for analysis of oxysterols, sterols, and fatty acid composition of phospholipids in the same biological sample. Total lipid extraction was determined after addition of several internal standards (epicoprostanol for sterols, 19-hydroxy-cholesterol for oxysterol and di-heptadecanoyl-phosphatidylcholine for phospholipid fatty acids). Cold acetone-mediated precipitation was then used to fractionate sterols from phospholipids. The phospholipid-containing precipitate was transmethylated for fatty acid analysis by gas chromatography. The sterol- and oxysterol-containing phase was saponified under mild conditions to avoid artificial oxysterol generation and was analyzed by gas chromatography after derivatization into trimethylsilyl ethers. The overall procedure was found to be specific with good recovery and reproducibility for sterols, oxysterols [mean coefficient of variation 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 treated-human low-density lipoproteins and erythrocytes. Results demonstrated that this method is a useful tool in assessing qualitative and quantitative differences in oxysterols and phospholipid fatty acid patterns attributed to lipid oxidation.