Disposition of flavonoids via enteric recycling: enzyme stability affects characterization of prunetin glucuronidation across species, organs, and UGT isoforms

Abstract

We characterized the in vitro glucuronidation of prunetin, a prodrug of genistein that is a highly active cancer prevention agent. Metabolism studies were conducted using expressed human UGT isoforms and microsomes/S9 fractions prepared from intestine and liver of rodents and humans. The results indicated that human intestinal microsomes were more efficient than liver microsomes in glucuronidating prunetin, but rates of metabolism were dependent on time of incubation at 37 degrees C. Human liver and intestinal microsomes mainly produced metabolite 1 (prunetin-5- O-glucuronide) and metabolite 2 (prunetin-4'- O-glucuronide), respectively. Using 12 human UGT isoforms, we showed that UGT1A7, UGT1A8, and UGT1A9 were mainly responsible for the formation of metabolite 1, whereas UGT1A1, UGT1A8, and UGT1A10 were mainly responsible for the formation of metabolite 2. This isoform-specific metabolism was consistent with earlier results obtained using human liver and intestinal microsomes, as the former (liver) is UGT1A9-rich whereas the latter is UGT1A10-rich. Surprisingly, we found that the thermostability of the microsomes was isoform- and organ-dependent. For example, human liver microsomal UGT activities were much more heat-stable (37 degrees C) than intestinal microsomal UGT activities, consistent with the finding that human UGT1A9 is much more thermostable than human UGT1A10 and UGT1A8. The organ-specific thermostability profiles were also evident in rat microsomes and mouse S9 fractions, even though human intestinal glucuronidation of prunetin differs significantly from rodent intestinal glucuronidation. In conclusion, prunetin glucuronidation is species-, organ-, and UGT-isoform-dependent, all of which may be impacted by the thermostability of specific UGT isoforms involved in the metabolism.

Fig. 1

Ultra performance LC (UPLC) chromatograms of prunetin and prunetin metabolites following UV absorbance (Fig.1A, Fig.1B, Fig.1C) or MS/MS spectral analysis (Fig.1D–1F). The UPLC methods were described in the “Method” section. Fig.1A shows the UPLC trace of 40 µM of prunetin metabolism by female rat jejunal microsome (FRJM), with peak of metabolite 1 (M1), metabolite 2 (M2), metabolite 3 (M3), internal standard (IS), and prunetin (PR) labeled. Fig.1B shows metabolites 1–3 (if present) of 40 µM of prunetin in female human jejunal microsomes (FHJM), female human liver microsomes (FHLM), female rat liver microsomes (FRLM), female rat jejunal microsomes (FRJM), expressed human UGT1A10 (UGT1A10), and expressed human UGT1A7 (UGT1A7). Notice the distinct pattern of metabolite formation. Fig.1C shows an alternative UPLC tracing using UV absorbance, which shows prunetin and its three glucuronidated metabolites. Fig.1D–1F shows the MS3 spectra of prunetin O-glucuronide (Fig.1D), prunetin C-glucuronide (Fig.1E) and prunetin (Fig.1F), respectively. The small windows in Fig.1D and Fig.1E show the MS2 full scan for the corresponding metabolite.

Fig. 2

Metabolism of prunetin by female human jejunal microsomes (FHJM), female human ileal microsomes (FHIM) and female human liver microsomes (FHLM) at two concentrations (40 or 2.5 µM) and two incubation time (8 and 24 hr). The experiments were conducted at 37°C for two incubation times and substrate concentrations and the amounts of metabolite 1 and metabolite 2 were measured using UPLC (Table 1). Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 3

Effects of concentration on the isoform-specific metabolism of prunetin. The experiments were conducted at 37°C for 1 hr and the amounts of metabolite 1 and metabolite 2 were measured using UPLC. Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 4

Effects of incubation time on the isoform-specific metabolism of prunetin at 40 µM. The experiments were conducted at 37°C for various times (1, 4, 8, or 24 hr) and the amounts of metabolite 1 and metabolite 2 were measured using UPLC. Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 5

Effects of incubation time on the isoform-specific metabolism of prunetin at 2.5 µM. The experiments were conducted at 37°C for various time and the amounts of metabolite 1 and metabolite 2 were measured using UPLC (Table 1). Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 6

Testing of thermal stability of UGT activities using human microsomes and expressed UGT microsomes. The abilities of UGT to form metabolite 1 or metabolite 2 were followed as a function of pre-incubation time (or the length of time where microsomes were incubated with everything but substrate). The concentration used was 40 µM and the actual reaction time was 1 hr. Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 7

Metabolism of prunetin by female rat jejunal microsomes (FRJM), and female rat liver microsomes (FRLM) at two concentrations (40 or 2.5 µM) and two incubation time (8 and 24 hr). The experiments were conducted at 37°C and the amounts of metabolite 1, metabolite 2 and metabolite 3 were measured using UPLC. Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 8

Total anion scan (TIC) or selective anion scan (m/z 441, m/z459, and m/z 283) of rat microsomal metabolism samples containing prunetin and three of its glucuronides before (Panel A) and after (Panel B) hydrolysis with β-D-glucuronidases (24 hrs). Clearly shown in this figure is that C-glucuronide (m/z 441) is not hydrolysable by β-D-glucuronidases. There was some cross talk between prunetin (m/z 283) and its glucuronides as these glucuronides are not very stable during the ionization process.

Fig. 9

Metabolism of prunetin by female mouse jejunal S9 fraction (FMS9F_SI), and female mouse liver S9 fraction (FMS9F_L) at two concentrations (40 or 2.5 µM) and two incubation time (8 and 24 hr). The experiments were conducted at 37°C and the amounts of metabolite 1 and metabolite 2 were measured using UPLC. Each bar is the average of three determinations and the error bars are the standard deviations of the mean.

Fig. 10

Metabolic pathways of prunetin in intestine and liver of rodents and humans. Position of C-glucuronide remained to be confirmed using NMR.