A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells.
A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. 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 inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification 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 quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells.
Robinet
Peggy
P
Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA.
Wang
Zeneng
Z
Hazen
Stanley L
SL
Smith
Jonathan D
JD
D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000302:isolation & purification / Q000378:metabolism*; D004798:Enzymes / Q000378:metabolism*; D005470:Fluorometry; D005487:Foam Cells / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D008264:Macrophages / Q000378:metabolism*; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry
D004798:Enzymes; D012997:Solvents; D002784:Cholesterol