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.
Highlights
Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed
We demonstrated that CE and FC could be quantitatively extracted from mouse arteries without tissue grinding and that lipid extraction simultaneously works for tissue fixation
Similar analysis performed on the extracts from the left and right common carotid arteries in the flow cessation model of accelerated atherosclerosis demonstrated that FC and CE preferentially accumulated in the ligated arteries 14 days after ligation (Fig. 7C and D)
Summary
Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. To enable novel approach of sequestial lipid and histological analysis, the mice were perfused with saline via the left ventricle, the arteries were dissected and placed into 1 ml of 2:1 chloroform/methanol solution.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
