Glucuronidation of flavonoids by recombinant UGT1A3 and UGT1A9
Introduction
More than 4000 chemically different flavonoids have been identified to date [1]. Some have been highlighted for their potential health-beneficial properties in in vitro experiments [2], while it is impossible to translate in vitro observations into the reality of the human situation without understanding the forms in which flavonoids and their metabolites circulate in vivo. In this way, the key point to determine in vivo pharmacological activities of flavonoids is their bioavailabilities and metabolisms within target tissues [2]. During absorption, flavonoids are extensively conjugated in small intestine and later in liver [3]. The conjugation mechanisms are so efficient that unchanged forms of flavonoids are generally either absent or present in low concentrations in blood [4]. It indicates that flavonoids exert their functions at least in part via conjugates.
Glucuronidation is one of the three major flavonoid conjugations [4] and catalyzed by UDP-glucuronosyltransferases (UGTs), the microsomal enzymes encoded by a multigene family in human. To date, four UGT families have been identified. UGT1A is the major subfamily responsible for xenobiotics glucuronidations. Over the years, liver was found to be the major tissue for UGT activities, while microsomes and recombinant enzymes from the intestine were also reported to be effective in vitro for glucuronidation of flavonoids [5], [6]. Hence the UGTs distributing in both liver and intestine, such as UGT1A3 [7] and UGT1A9, may be more important in flavonoid glucuronidation than other UGTs.
In the present study, catalyzing activities of UGT1A3 and UGT1A9, recombinantly expressed in Bac-to-Bac insect cell, to nineteen flavonoids were tested. Several new substrates were reported, and even more important, metabolism characteristics of UGT1A3 and UGT1A9 on flavonoids were presented.
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Materials
Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Restriction endonucleases, DNA molecular marker and T4 ligase were obtained from MBI Fermentas (Amherst, NY, USA). Agarose LE was supplied by Roche Diagnostics (Mannheim, Germany). UDP-glucuronic acid, alamethicin (dissolved in methanol), d-saccharic acid 1,4-lactone, and β-glucuronidase from E. coli were provided by Sigma (St. Louis, MO, USA). Quercetin, isorhamnetin, baicalin, luteolin, puerarin, kaempferol, rutin,
Glucuronidation assay
In the present study, a series of flavonoids was investigated for glucuronidation reactivities with recombinant UGT1A3 or UGT1A9. Among nineteen tested flavonoids, 11 compounds were catalyzed by recombinant UGT1A3 or UGT1A9. For example, luteolin (Fig. 3a and a′) or apigenin (Fig. 3b and b′), two tested flavones, were catalyzed into three metabolites or one metabolite. The representative chromatograms are shown in Fig. 3. All hypothesized glucuronide peaks disappeared after β-glucuronidase
Discussion
Flavonoids have attracted increasing attention in the past several decades for their great abundance and antioxidant properties [15], [16], [17]. Some researches pointed out that flavonoids were generally recovered in plasma and urine as conjugated derivatives, with no or trace amounts of native forms detected [18], [19], [20], [21], [22]. As one of the three major conjugation pathways, glucuronidation plays an important role in biological effects of flavonoids. In the past, many studies on the
Acknowledgements
This project was supported by the National Natural Science Foundation of China (30701038), the Specialized Research Fund for the Doctoral Program of Higher Education of China (2004335013) and Zhejiang Provincial Key Science and Technology Foundation (2005C13026).
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