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 determin ation of the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma. Thi s method is based on a stable isotope-dilution technique by gas chroma tography-selected-ion monitoring mass spectrometry. 7 alpha-Hydroxy-4- cholesten-3-one was extracted from plasma by salting-out extraction, a nd then purified by serial solid-phase extractions. The extract was tr eated with O-methylhydroxylamine hydrochloride and then dimethylethyls ilylated. The resulting methyloxime-dimethylethylsilyl ether derivativ e was quantified by gas chromatography-selected-ion monitoring mass sp ectrometry with a high-resolution mode. The plasma levels of 7 alpha-h ydroxy-4-cholesten-3-one were correlated with the cholesterol 7 alpha- hydroxylase activity to a higher degree than those of any other form o f 7 alpha-hydroxycholesterol (r = 0.84, n = 16, p <0.0001). The presen t method was applied to monitor the circadian rhythm of 7 alpha-hydrox y-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 fo r the monitoring of bile acid biosynthesis in the human liver.