Separation and characterization of carboxyl-linked glucuronides of bile acids in incubation mixture of rat liver microsomes.
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 alpha- and beta-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.
Goto
J
J
Faculty of Pharmaceutical Sciences, Tohoku University Aobayama, Sendai, Japan.
Murao
N
N
Nakada
C
C
Motoyama
T
T
Oohashi
J
J
Yanagihara
T
T
Niwa
T
T
Ikegawa
S
S
D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002853:Chromatography, Liquid; D005965:Glucuronates / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000737:chemistry* / Q000201:enzymology; D051381:Rats
D001647:Bile Acids and Salts; D002264:Carboxylic Acids; D005965:Glucuronates; D014453:Glucuronosyltransferase