Glucuronides Hydrolysis by Intestinal Microbial β-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species.

Christabel Ebuzoeme,Imoh Etim,Jamie Song,Ting Du,Autumn Ikimi,Ming Hu,Song Gao,Dong Liang

doi:10.3390/pharmaceutics13071043

Christabel Ebuzoeme, Imoh Etim + Show 6 more

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https://doi.org/10.3390/pharmaceutics13071043

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Abstract

Glucuronides hydrolysis by intestinal microbial β-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69–1.32-fold, 2.9–12.9-fold, and 0.28–1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 μmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 μM for mouse, rat, and human, respectively. The inter-individual difference was significant (4–6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant.

Highlights

The role of gut microflora in health has gained increasing attention in the past decades [1,2,3]
The results showed that the relative hydrolysis rates for probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) were magnesium ions
The results showed that, for rats and mice, the relative hydrolysis rates of pNPG were highest at pH 6.5 and for humans the highest rate was at pH 7.4 (Figure 6)

Summary

Introduction

The role of gut microflora in health has gained increasing attention in the past decades [1,2,3]. One of the major benefits from intestinal microflora is that the commensal bacteria could generate beneficial metabolites, such as cancer-preventive compound equol for daidzein and compound k from ginsenosides, for the host from dietary components through different biotransformation pathways [4,5,6]. These metabolites either accumulate in the gastrointestinal system or reach distant organs, which are associated with certain physiological or pathological effects (e.g., gastrointestinal inflammation and carcinogenesis [7]).

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