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

A number of oxysterols have been implicated in metabolic regulation, Key am ong these are (24S),25-epoxy-cholesterol and (24S)-hydroxycholesterol, high affinity ligands for the nuclear transcription factor liver X receptor alp ha; 27-hydroxycholesterol, a bile acid synthetic intermediate; and 25-hydro xycholesterol, which has been used to study regulation of lipid metabolism by the sterol regulatory element-binding protein family of transcription fa ctors. Investigation of the physiological importance of these compounds in vivo has been hampered by lack of analytical methods to reproducibly and ac curately determine their concentrations in tissues. This article describes a method designed to determine quantitatively the amounts of these importan t sidechain oxysterols by derivatization to the Delta (4)-3-ketones followe d 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 ha ve distinct subcellular distributions and responses to physiological pertur bations in vivo.