title: Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue.
abstract: Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.
authors: Department of Medicinal Analytical Chemistry, Lilly Research Laboratories, Indianapolis, IN 46285, USA.
authors: Ming-Shang
authors: MS
authors: Kuo
authors: J Michael
authors: JM
authors: Kalbfleisch
authors: Pamela
authors: P
authors: Rutherford
authors: Donetta
authors: D
authors: Gifford-Moore
authors: Xiao-di
authors: XD
authors: Huang
authors: Robert
authors: R
authors: Christie
authors: Kwan
authors: K
authors: Hui
authors: Kenneth
authors: K
authors: Gould
authors: Mark
authors: M
authors: Rekhter
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D012015:Reference Standards; D053719:Tandem Mass Spectrometry
chemical_list: D002784:Cholesterol