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

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 β-D-glucuronopyranosyl-(1→2)-β-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.