Separation and characterization of carboxyl-linked glucuronides of bile acids in incubation mixture of rat liver microsomes

Junichi Goto, Naoaki Murao, Chifumi Nakada, Takashi Motoyama, Junji Oohashi, Takamitsu Yanagihara, Toshifumi Niwa, Shigeo Ikegawa

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)

Abstract

The carboxyl-linked 24-glucuronides of common bile acids have been identified by means of liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) in an incubation mixture with a male Wistar rat liver microsomal fraction. The authentic specimens of bile acid 24-glucuronide acetate-methyl esters were synthesized unequivocally using the Mitsunobu reaction, and the APCI-mass spectrometric properties of these glucuronide derivatives were also characterized. After incubation of common unconjugated bile acids with hepatic microsomes, glucuronides were extracted and purified with a Sep-Pak C18 cartridge and lipophilic ion exchange gel, piperidino-hydroxypropyl Sephadex LH-20, and then derivatized into the acetate-methyl esters. Subsequent resolution into α- and β- isomers at the glucuronosyl linkage was attained by LC on Cosmosil 5C8 and Sumichiral OA-2500 columns using 200 mM ammonium acetate (pH 7.0)-methanol (1:4, v/v), where 24-glucuronides were monitored with characteristic positive ions [M + NH4]+. The 24-glucuronides of lithocholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acid were definitely characterized, in contrast to no formation of corresponding 3-glucuronides.

Original languageEnglish
Pages (from-to)186-192
Number of pages7
JournalSteroids
Volume63
Issue number4
DOIs
Publication statusPublished - 1998 Apr

Keywords

  • Atmospheric pressure chemical ionization
  • Bile acid
  • Glucuronide
  • Glucuronosyltransferase
  • LC/MS

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

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