Determination of sterols, oxysterols, and fatty acids of phospholipids in cells and lipoproteins: A one-sample method |
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Authors: | D Blache P Durand F Girodon L Gesquière N Loreau |
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Affiliation: | (1) NSERM U498, Laboratoire de Biochimie des Lipoprotéines, Faculté de Médecine, Université de Bourgogne, Dijon, France;(2) Laboratoire d’Hématologie, Centre Hospitalier Régional Universitaire, Dijon, France |
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Abstract: | 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. |
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Keywords: | Cholesterol oxides erythrocytes free radicals low-density lipoproteins oxidation oxysterols phospholipid fatty acids |
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