Determination of 7α-hydroxy-4-cholesten-3-one level in plasma using isotope-dilution mass spectrometry and monitoring its circadian rhythm in human as an index of bile acid biosynthesis

Abstract

A highly sensitive and specific method has been developed for determination of the level of 7α-hydroxy-4-cholesten-3-one in plasma. This method is based on a stable isotope-dilution technique by gas chromatography—selected-ion monitoring mass spectrometry. 7α-Hydroxy-4-cholesten-3-one was extracted from plasma by salting-out extraction, and then purified by serial solid-phase extractions. The extract was treated with O-methylhydroxylamine hydrochloride and then dimethylethylsilylated. The resulting methyloxime—dimethylethylsilyl ether derivative was quantified by gas chromatography—selected-ion monitoring mass spectrometry with a high-resolution mode. The plasma levels of 7α-hydroxy-4-cholesten-3-one were correlated with the cholesterol 7α-hydroxylase activity to a higher degree than those of any other form of 7α-hydroxycholesterol ( r = 0.84, n = 16, p < 0.0001). The present method was applied to monitor the circadian rhythm of 7α-hydroxy-4-cholesten-3-one levels in human plasma. It was concluded that the plasma level of 7α-hydroxy-4-cholesten-3-one is a useful index for the monitoring of bile acid biosynthesis in the human liver.