Analysis of free radical production capacity in mouse faeces and its possible application in evaluating the intestinal environment: a pilot study

Yoshihisa Wakita,Asako Saiki,Hirotaka Kaneda,Susumu Okamoto,Youichi Tsuchiya,Shuichi Segawa,Hiromi Kameya

doi:10.1038/s41598-019-56004-x

Abstract

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome. Oxygen free radicals such as superoxide and the hydroxyl radical (•OH) are generated during normal cellular metabolism. They are toxic to both eukaryotic and prokaryotic cells and might thus affect intestinal homeostasis. However, the effect of oxygen free radicals on the intestinal environment has not been widely studied. Herein, we applied electron spin resonance spectroscopy with spin trapping reagents to evaluate oxygen free radical production capacity in the intestinal lumen and the faeces of mice. •OH was generated in faeces and lumens of the small and large intestines. There were no remarkable differences in •OH levels between faeces and the large intestine, suggesting that faeces can be used as alternative samples to estimate the •OH production capacity in the colonic contents. We then compared free radical levels in faecal samples among five different mouse strains (ddY, ICR, C57BL/6, C3H/HeJ, and BALB/c) and found that strain ddY had considerably higher levels than the other four strains. In addition, strain ddY was more susceptible to dextran sulphate sodium-induced colitis. These differences were possibly related to the relative abundance of the gut bacterial group Candidatus Arthromitus, which is known to modulate the host immune response. From these results, we suggest that the production capacity of oxygen free radicals in mouse faeces is associated with intestinal homeostasis.

Highlights

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome
To evaluate the capacity of oxygen free radical production in the lumens of the large and small intestines, we performed a ligated intestinal loop experiment (Fig. 1a) and oxygen free radicals were detected by the Electron spin resonance (ESR) spin-trapping technique (Fig. 1b)
Methane sulfinic acid, which is a byproduct of the reaction between DMSO and OH, have www.nature.com/scientificreports been routinely analyzed as a proxy of the OH production capacity in faeces

Summary

Introduction

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome. O2− and OH are highly oxidizing species with a reduction potential of ~1.6 V and ~2.6–2.7 V, respectively[15]. These oxygen free radicals instantly react with almost all biomolecules, and they can only be trapped by a spin trap, provided that the spin trap is in close proximity to the radicals at the exact moment when they are generated. We applied ESR with spin trapping reagents to assess free radical production capacity in the intestinal lumen and faeces of mice. A possible relationship between this faecal free radical production capacity and the intestinal microbiome and/or intestinal homeostasis is discussed

Methods

Results

Conclusion