Combination of an enzymatic method and HPLC for the quantitation of cholesterol in cultured cells.
The study of the cellular events that lead to the foam cell formation requires the development of fast, accurate, and sensitive methods to quantify cholesterol in cultured cells. Here we describe a procedure that allows the rapid determination of free and total cholesterol in a reduced number of cells, which makes it very suitable for cholesterol determination in cell cultures. The method consists of the enzymatic conversion of cholesterol to cholest-4-ene-3-one by cholesterol oxidase followed by the analysis of the sample by high performance liquid chromatography (HPLC) to detect this oxidized product. Due to the relatively high wavelength at which cholest-4-ene-3-one has its maximum absorption (240 nm), other cellular components do not interfere with the chromatographic procedure and prior lipid extraction is not required. Moreover, the duration of each chromatogram is about 3 min, contributing to the celerity of the method. All the cholesteryl esters used (oleate, palmitate, stearate and linoleate) were quantitatively hydrolyzed by incubation with cholesterol esterase; this was observed to occur with both pure standards and in cell homogenates. Sensitivity is enough to allow the determination of free and total cholesterol in less than 5 x 10(3) cells. We have applied this method to human monocyte-derived macrophages and the values obtained for free and total cholesterol are in close agreement with published data.
Contreras
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Unidad de Dislipemias, Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Castro
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M
Bocos
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C
Herrera
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Lasunción
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MA
D002478:Cells, Cultured; D002783:Cholestenones / Q000032:analysis; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D009000:Monocytes / Q000737:chemistry*
D002783:Cholestenones; D002788:Cholesterol Esters; D007202:Indicators and Reagents; C000475:cholest-4-en-3-one; D002784:Cholesterol; D002789:Cholesterol Oxidase