A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

Abstract

This article is available online at http://www.jlr.org levels can be separated into two groups: 1 ) analytical methods such as gas-liquid chromatography or liquid chromatography coupled with fl ame ionization or mass spectrometry detection and quantifi cation ( 2-4 ); and 2 ) enzymatic assays, which can be colorimetric ( 5, 6 ) or fl uorometric ( 7 ). However, the chromatographic and mass spectrometry methods require specialized equipment and chemical saponifi cation of cholesterol esters; furthermore, these methods are time consuming and require processing samples one at a time. In contrast, the colorimetric and fl uorometric enzymatic methods are simple, sensitive, and relatively fast assays that can process many samples at once. The principal of a sensitive fl uorometric assay for the measurement of total cholesterol is shown below, with free cholesterol measured by omitting the fi rst esterase step, and cholesterol mass in cholesterol esters calculated as the difference between the total and free cholesterol contents. HRPThe enzymatic method is the principal method used for measuring cholesterol levels in plasma, where cholesterol is solubilized in an aqueous solution through its incorporation in lipoproteins. However, in order to accurately measure the cholesterol content of cells or tissue by this method, one must identify a suitable solvent that allows good recovery of free and esterifi ed cholesterol in solution and that is compatible with the enzymes and substrates. Previously, the use of the enzymatic cholesterol assay for cells and tissues has been limited due to the inadequate quantitative recovery of free cholesterol and cholesterol esters in enzyme compatible solvents; in fact, a methods paper has argued that the enzymatic method cannot be used for this purpose due to poor extraction and solubiliAbstract A precise and sensitive method for measuring cellular free and esterifi ed cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and effl ux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to ineffi cient solubilization of total cholesterol in enzyme compatible solvents. We present an effi cient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponifi cation or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantifi cation in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusio...

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