Formation of a Structurally Novel, Serial Diglucuronide of 4-Hydroxybiphenyl by Further Glucuronidation of a Monoglucuronide in Dog Liver Microsomes

Abstract

Incubation of 4-hydroxybiphenyl (p-phenylphenol) in the presence of UDP-glucuronic acid (UDPGA) with liver microsomes from male and female dogs produced a more polar metabolite peak than a simultaneously produced peak of 4-hydroxybiphenyl monoglucuronide in the high performance liquid chromatography (HPLC) chromatogram. Tandem mass spectrometry (MS/MS) and two-dimensional nuclear magnetic resonance (NMR) analyses revealed this polar metabolite as a 4-hydroxybiphenyl diglucuronide having a beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl moiety, where the two glucuronic acids are connected directly at the 1''-->2' position. Liver microsomes from Sprague-Dawley rat, cynomolgus monkey and human, converted 4-hydroxybiphenyl only to the monoglucuronide, suggesting that there is a dog UDP-glucuronosyltransferase (UGT), with a wider substrate specificity capable of glucuronidating 4-hydroxybiphenyl monoglucuronide to the diglucuronide.

Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs

“…4), which is common among the diglucuronides characterized previously (Dixon et al, 1989;Murai et al, 2002Murai et al, , 2005Murai et al, , and 2007). The hydroxyl group at the C2Ј position of the glucuronosyl group in a specific monoglucuronide was recognized as a functional group for further glucuronidation by unique dog UGT isoforms, which would have similar enzymatic properties to human UGT1A8, as demonstrated in our previous study in vitro (Murai et al, 2006).…”

“…After the discovery of nalmefene diglucuronide, we reported that diglucuronides could be produced in vitro from 4-hydroxybiphenyl, a phenolic compound used commonly as a model substrate for glucuronidation (Murai et al, 2002). Moreover, we found that endogenous sex steroids, namely androsterone, dihydrotestosterone (DHT), estradiol, estriol, estrone, and testosterone, also undergo diglucuronidation in vitro (Murai et al, 2005).…”

Human Udp-Glucuronosyltransferase, Ugt1a8, Glucuronidates Dihydrotestosterone to a Monoglucuronide and Further to a Structurally Novel Diglucuronide

“…Pooled rat liver microsomes were also obtained from BD Gentest. Dog liver microsomes used were prepared as described previously (Murai et al, 2002). All microsomes were stored frozen at approximately Ϫ80°C until use.…”

“…After the discovery of nalmefene diglucuronide in vivo, we were the first to report the in vitro production of the diglucuronide of 4-hydroxybiphenyl, a nonplanar phenolic compound used commonly as a model substrate for glucuronidation, using dog liver microsomes (Murai et al, 2002). The diglucuronidation reaction was found to proceed in a stepwise manner, from 4-hydroxybiphenyl to 4-hydroxybiphenyl monoglucuronide and from the monoglucuronide to the diglucuronide.…”

“…The diglucuronidation reaction was found to proceed in a stepwise manner, from 4-hydroxybiphenyl to 4-hydroxybiphenyl monoglucuronide and from the monoglucuronide to the diglucuronide. Rat, monkey, and human liver microsomes showed undetectable 4-hydroxybiphenyl diglucuronidation activities (Murai et al, 2002). Although the formation of this type of diglucuronide had been found solely for xenobiotics, we recently found that physiologically important endogenous sex steroids, namely, androsterone, dihydrotestosterone (DHT), estradiol, estriol, estrone, and testosterone, also undergo diglucuronidation in vitro (Murai et al, 2005).…”