Abstract

Glucuronidation, which may take place on the phenolic hydroxyl and carboxyl groups, is a major pathway of metabolism for thyroxine (T4) and triiodothyronine (T3). In this study, a liquid chromatography/mass spectrometry (LC/MS) method was developed to separate phenolic and acyl glucuronides of T4 and T3. The method was used to collect the phenolic glucuronide of T4 for definitive characterization by NMR and to determine effects of incubation pH, species differences, and human UDP-glucuronosyltransferases (UGTs) involved in the formation of the glucuronides. Formation of T4 phenolic glucuronide was favored at pH 7.4, whereas formation of T4 acyl glucuronide was favored at pH 6.8. All the UGTs examined catalyzed the formation of T4 phenolic glucuronide except UGT1A4; the highest activity was detected with UGT1A3, UGT1A8, and UGT1A10, followed by UGT1A1 and UGT2B4. Formation of T3 phenolic glucuronide was observed in the order of UGT1A8 > UGT1A10 > UGT1A3 > UGT1A1; trace activity was observed with UGT1A6 and UGT1A9. UGT1A3 was the major isoform catalyzing the formation of T4 and T3 acyl glucuronides. In liver microsomes, phenolic glucuronidation was the highest in mice for T4 and in rats for T3 and lowest in monkeys for both T4 and T3. Acyl glucuronidation was highest in humans and lowest in mice for T4 and T3. Phenolic glucuronidation was higher than acyl glucuronidation for T4 in humans; in contrast, the acyl glucuronidation was slightly higher than phenolic glucuronidation for T3. UGT activities were lower toward T3 than T4 in all the species. The LC/MS method was a useful tool in studying glucuronidation of T4 and T3.