DETERMINATION OF 7-ALPHA-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

A highly sensitive and specific method has been developed for determination of the level of 7 alpha-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. 7 alpha-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 7 alpha-hydroxy-4-cholesten-3-one were correlated with the cholesterol 7 alpha-hydroxylase activity to a higher degree than those of any other form of 7 alpha-hydroxycholesterol (r = 0.84, n = 16, p <0.0001). The present method was applied to monitor the circadian rhythm of 7 alpha-hydroxy-4-cholesten-3-one levels in human plasma. It was concluded that the plasma level of 7 alpha-hydroxy-4-cholesten-3-one is a useful index for the monitoring of bile acid biosynthesis in the human liver.