title: Isolation and identification of diglucuronides of some endogenous steroids in dogs.
abstract: Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C(19) steroids, including M1 (C(31)H(46)O(14)) and M2 (C(31)H(44)O(14)), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan. murai.takahiro.a4@daiichisankyo.co.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Murai
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Yamamura
authors.forename: Takashi
authors.initials: T
authors.lastname: Nitanai
authors.forename: Naozumi
authors.initials: N
authors.lastname: Samata
authors.forename: Makoto
authors.initials: M
authors.lastname: Takei
authors.forename: Haruo
authors.initials: H
authors.lastname: Iwabuchi
authors.forename: Kohji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Kei
authors.initials: K
authors.lastname: Mikamoto
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D001646:Bile / Q000737:chemistry; D004285:Dogs; D020719:Glucuronides / Q000378:metabolism* / Q000652:urine; D006801:Humans; D008252:Macaca fascicularis; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D052616:Solid Phase Extraction; D045165:Testosterone Congeners / Q000378:metabolism*
chemical_list: D020719:Glucuronides; D045165:Testosterone Congeners