Identification of 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid in serum from patients treated with ursodeoxycholic acid

An unknown bile acid was found by gas-liquid chromatography in the serum of patients who were administered ursodeoxycholic acid for the treatment of cholesterol gallstones. Identification of the chemical structure of the unknown bile acid was performed by the use of gas-liquid chromatography-mass spectrometry. Mass spectrum analysis of the methyl ester trimethylsilyl ether of the bile acid showed explicitly that this is dihydroxy-5 beta-cholanoic acid, since peaks at m/e 460 and 370 characteristic of methyl ester trimethylsilyl ether of dihydroxy bile acid were clearly exhibited. Sites of the two hydroxyl groups on the steroid nucleus were determined to be at the 3- and 7-positions by conversion of the bile acid to the corresponding dioxo-cholanoic acid and by comparison of the gas-liquid chromatographic behavior with those of authentic dioxo bile acids. Four authentic 3,7-dihydroxy-5 beta-cholan-24-oic acids were chemically synthesized and retention times and mass spectra of their methyl ester trimethylsilyl ether derivatives compared precisely with that of the unknown bile acid. The results indicate that the unknown bile acid is 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid. Preliminary experiments suggest that 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid is absent as amino acid-conjugated forms in serum. It is also suggested that the bile acid is excreted into urine but not into bile.