Analysis of cholesterol and desmosterol in cultured cells without organic solvent extraction |
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Authors: | Edward H Goh Debra K Krauth Scott M Colles |
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Affiliation: | (1) Medical Sciences Program, Indiana University, School of Medicine, 47405 Bloomington, Indiana |
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Abstract: | Cultured cell sterols such as cholesterol and desmosterol are usually extracted into organic solvents before they are quantified
with cholesterol esterase and oxidase. A method to quantify these cultured cell sterols using cholesterol enzymes without
prior organic solvent extraction is described. In this method, a suspension or monolayer of cultured L-M, U-937, or PC-12
cells is digested with 0.1% sodium dodecyl sulfate (SDS), and the digest treated with microbial cholesterol enzymes. The quantity
of oxidized sterols produced by the reaction can be measured easily with high-pressure liquid chromatography, when a mixture
of sterols is present, or by the production of hydrogen peroxide when only one sterol is present. This method is easier and
safer to use than solvent extraction and can greatly expedite the quantitation of cultured cell sterols. Preliminary data
show that other lipids such as choline phospholipids, triglycerides, and fatty acids can also be directly quantified in SDS
cell digest by using specific enzymes to transform these lipids into hydrogen peroxides. |
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