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Originally published In Press as doi:10.1194/jlr.D700037-JLR200 on March 18, 2008

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Journal of Lipid Research, Vol. 49, 1353-1363, June 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Ming-Shang Kuo*, J. Michael Kalbfleisch*, Pamela Rutherford{dagger}, Donetta Gifford-Moore{dagger}, Xiao-di Huang{dagger}, Robert Christie{dagger}, Kwan Hui{dagger}, Kenneth Gould{dagger} and Mark Rekhter1,{dagger}

* Departments of Medicinal Analytical Chemistry, Lilly Research Laboratories, Indianapolis, IN 46285
{dagger} Atherosclerosis and Metabolic Syndrome Department, Lilly Research Laboratories, Indianapolis, IN 46285

Published, JLR Papers in Press, March 18, 2008.

1 To whom correspondence should be addressed. e-mail: 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. 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 PPAR{alpha},{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.

Supplementary key words quantification • liquid chromatography • mass spectrometry • atherosclerosis • animal models • transgenic mice • immunohistochemistry


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