| A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells. | |
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MedLine Citation:
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PMID: 20688754 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Peggy Robinet; Zeneng Wang; Stanley L Hazen; Jonathan D Smith |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2010-08-04 |
Journal Detail:
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Title: Journal of lipid research Volume: 51 ISSN: 0022-2275 ISO Abbreviation: J. Lipid Res. Publication Date: 2010 Nov |
Date Detail:
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Created Date: 2010-10-13 Completed Date: 2011-01-19 Revised Date: 2011-11-01 |
Medline Journal Info:
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Nlm Unique ID: 0376606 Medline TA: J Lipid Res Country: United States |
Other Details:
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Languages: eng Pagination: 3364-9 Citation Subset: IM |
Affiliation:
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Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cattle Cholesterol / isolation & purification, metabolism* Enzymes / metabolism* Fluorometry Foam Cells / metabolism* Gas Chromatography-Mass Spectrometry Macrophages / metabolism* Reproducibility of Results Solvents / chemistry |
| Grant Support | |
ID/Acronym/Agency:
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P01 HL076491/HL/NHLBI NIH HHS; P01 HL098055/HL/NHLBI NIH HHS; P01 HL098055-01A17172/HL/NHLBI NIH HHS; R01 HL066082/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Enzymes; 0/Solvents; 57-88-5/Cholesterol |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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