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 C19 steroids, including M1 (C31H46O14) and M2 (C31H44O14), 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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019059.
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ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, uridine 5′-diphosphoglucuronic acid; NMR, nuclear magnetic resonance; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; CID, collision-induced dissociation; HSQC, heteronuclear single quantum correlation spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; DMSO, dimethyl sulfoxide.
- Received October 12, 2007.
- Accepted November 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs
