Repeated Glucuronidation at One Hydroxyl Group Leads to Structurally Novel Diglucuronides of Steroid Sex Hormones

Abstract

Androgens (androsterone, dihydrotestosterone and testosterone) and estrogens (estradiol, estriol and estrone) were incubated with liver microsomes from rats, dogs, monkeys and humans in the presence of uridine diphosphoglucuronic acid (UDPGA), and the glucuronides produced were structurally characterized by liquid chromatography-tandem mass spectrometry. After 2-h incubation with dog liver microsomes, all substrates tested were converted (approximately 2-10%) to structurally novel diglucuronides, where two glucuronosyl groups are bound to a single hydroxyl group in tandem. Two-dimensional nuclear magnetic resonance spectroscopy unambiguously elucidated the chemical structures of the 3-O-diglucuronide of estrone and the 17-O-diglucuronide of testosterone isolated from the incubation mixture. Monkey and human liver microsomes were also found to have the activity to form this type of diglucuronide, albeit more slowly than the dog liver microsomes, but rat liver microsomes produced no detectable diglucuronides. The rate of formation of estrone 3-O-diglucuronide from the corresponding monoglucuronide in dog liver microsomes followed classical Michaelis-Menten kinetics at substrate concentrations from 50 to 1000 microM, with a K(m) value of 127.1 microM and a V(max) value of 47.0 pmol/min/mg protein.

Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs

“…Significant species differences were observed in the production of diglucuronides in vitro. Dog liver microsomes exhibited the highest activity for diglucuronidation, whereas monkey and human liver microsomes exhibited detectable diglucuronidation activity for particular steroids (Murai et al, 2005). Activity producing DHT diglucuronide from the corresponding monoglucuronide in recombinant human UGT iso-forms was found in UGT1A8, UGT1A1, and UGT1A9, in descending order (Murai et al, 2006).…”

“…The fact that androgens and estrogens are good substrates for diglucuronidation in vitro using dog liver microsomes (Murai et al, 2005) does not necessarily indicate that the diglucuronidation reaction is occurring under in vivo conditions. To date, no diglucuronides produced from endogenous compounds have been identified in vivo.…”

“…However, when the glucuronidation does not supply enough hydrophilicity to a molecule or when the glucuronide is still recognized as a good substrate by UGTs, subsequent glucuronidation may occasionally take place on the glucuronides. Glucuronides carrying two glucuronosyl moieties are classified into two groups, namely bisglucuronides and diglucuronides (Murai et al, 2005). Bisglucuronides are defined as glucuronides where two separate functional groups on the parent molecule are conjugated at the same time.…”

“…Bisglucuronides are relatively common phase II metabolites of both endogenous and exogenous compounds (Gordon et al, 1976;Bock et al, 1992). On the other hand, diglucuronides are defined as particular glucuronides where a single functional group on the parent molecule is repeatedly conjugated by two glucuronosyl groups in tandem (Murai et al, 2005).…”

“…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). Significant species differences were observed in the production of diglucuronides in vitro.…”