Glucuronidation of macelignan by human liver microsomes and expressed UGT enzymes: identification of UGT1A1 and 2B7 as the main contributing enzymes

Abstract

Macelignan is a natural phenolic compound that possesses many types of health benefits such as antiinflammation. This study aimed to characterize the metabolism of macelignan via the glucuronidation pathway and to identify the main UGT enzymes involved in macelignan glucuronidation. The rates of glucuronidation were determined by incubating macelignan with UDPGA-supplemented microsomes. Kinetic parameters were derived by fitting an appropriate model to the data. Reaction phenotyping, the relative activity factor (RAF) approach and activity correlation analysis were employed to identify the main UGT enzymes contributing to the hepatic metabolism of macelignan. Glucuronidation of macelignan in pooled human liver microsomes (pHLM) was rather efficient with a high CLint (the intrinsic clearance) value of 13.90 ml/min/mg. All UGT enzymes, except UGT1A4, 1A6 and 2B10, showed metabolic activities toward macelignan. UGT1A1 and 2B7 were the enzymes with the highest activities; the CLint values were 4.92 and 2.13 ml/min/mg, respectively. Further, macelignan glucuronidation was significantly correlated with 3-O-glucuronidation of β-estradiol (r = 0.69; p < 0.01) and glucuronidation of zidovudine (r = 0.60; p < 0.05) in a bank of individual HLMs (n = 14). Based on the RAF approach, UGT1A1 and 2B7, respectively, contributed 55.40% and 32.20% of macelignan glucuronidation in pHLM. In conclusion, macelignan was efficiently metabolized via the glucuronidation pathway. It was also shown that UGT1A1 and 2B7 were probably the main contributors to the hepatic glucuronidation of macelignan.

Decreased Expression of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Leads to Reduced Glucuronidation of Flavonoids in UGT1A1-Overexpressing HeLa Cells: The Role of Futile Recycling

“…Apigenin-7-O-glucuronide (or apigenin glucuronide (AG)) and genistein-7-O-glucuronide (or genistein glucuronide (GG)) were synthesized in our laboratory using rat liver microsomes as described. 28 Cell Culture. HeLa1A1 cells and MRP4 knock-down HeLa1A1 cells (referred to as HeLa1A1-MRP4-shRNA cells) were seeded into six-well plates at a density of 4.0 × 10 5 cells/well and were grown using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).…”

Regio- and Isoform-Specific Glucuronidation of Psoralidin: Evaluation of 3-O-Glucuronidation as a Functional Marker for UGT1A9

“…The glucuronidation assay was performed as described in our previous publications . The stock solutions of aglycones were prepared in methanol–dimethyl sulfoxide (DMSO) (4:1, v/v) and diluted with methanol–DMSO (4:1, v/v) to the desired concentrations immediately before use.…”

“…A large amount of psoralidin glucuronides were generated using HLMs according to the glucuronidation assay protocol. The glucuronides were isolated and purified as described . In brief, the incubation mixture after reaction was concentrated and injected into the Dionex U300 HPLC system.…”

“…Structural identification of psoralidin glucuronides (G1 and G2) were performed using the UPLC‐quadrupole time‐of‐flight/MS method as described . Chromatographic separation was performed using the Waters ACQUITY UPLC system and BEH column (2.1 × 50 mm, 1.7 μm; Waters, Milford, Massachusetts).…”

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound

“…Bakuchiol was incubated with liver microsomes or expressed UGT enzymes to determine the rates of glucuronidation as described in our previous publication (Liu et al, 2014a). In brief, the incubation medium contained the enzyme preparation, MgCl 2 (0.88 mM), saccharolactone (4.4 mM), alamethicin (22 mg/ml), UDPGA (3.5 mM) and bakuchiol in 50 mM potassium phosphate (pH 7.4).…”