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Journal of Lipid Research, Vol. 42, 649-658, April 2001
Copyright © 2001 by Lipid Research, Inc.

Methods

Key regulatory oxysterols in liver: analysis as ⁴-3-ketone derivatives by HPLC and response to physiological perturbations

Correspondence to: Thomas A. Spencer, To whom correspondence should be addressed., taspen{at}dartmouth.edu (E-mail)

A number of oxysterols have been implicated in metabolic regulation. Key among these are (24S),25-epoxycholesterol and (24S)-hydroxycholesterol, high affinity ligands for the nuclear transcription factor liver X receptor ; 27-hydroxycholesterol, a bile acid synthetic intermediate; and 25-hydroxycholesterol, which has been used to study regulation of lipid metabolism by the sterol regulatory element-binding protein family of transcription factors. Investigation of the physiological importance of these compounds in vivo has been hampered by lack of analytical methods to reproducibly and accurately determine their concentrations in tissues.

This article describes a method designed to determine quantitatively the amounts of these important side-chain oxysterols by derivatization to the ⁴-3-ketones followed by high performance liquid chromatography. The method was validated with known standards and then was used to determine the concentrations of these oxysterols in rodent liver under various physiological conditions. All four oxysterols were present in the picogram per milligram protein range and have distinct subcellular distributions and responses to physiological perturbations in vivo. — Zhang, Z., D. Li, D. E. Blanchard, S. R. Lear, S. K. Erickson, and T. A. Spencer. Key regulatory oxysterols in liver: analysis as ⁴-3-ketone derivatives by HPLC and response to physiological perturbations. J. Lipid Res. 2001. 42: 649;–658.

Supplementary key words: (24S),25-epoxycholesterol, (24S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, synthesis, HPLC analysis, mass spectrometry

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