Submitted on November 7, 2007
Revised on March 13, 2008
Accepted on March 18, 2008
Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue
Ming-Shang Kuo, J Michael Kalbfleisch, Pamela Rutherford, Donetta Gifford-Moore, Xiao-Di Huang, Robert Christie, Kwan Hui, Kenneth Gould, and Mark Rekhter
Eli Lilly and Co, Indianapolis, IN 46285
Corresponding Author: rekhter_mark{at}lilly.com
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. De-lipidated tissues can be embedded in paraffin, sectioned and stained. Microscopic images obtained from de-lipidated arteries have not revealed any structural alterations. De-lipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE-/- mice, LC/MS/MS revealed early anti-atherosclerotic effects of dual PPAR agonist LY465606 in both brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in cholesterol ester and free cholesterol 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 up by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.
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