INFLAMMATORY BOWEL DISEASE

Mucosal sulfhydryl compounds evaluation by in vivo electron spin resonance spectroscopy in mice with experimental colitis

H Togashi¹, K Oikawa², T Adachi¹, K Sugahara¹, J Ito¹, T Takeda¹, H Watanabe¹, K Saito¹, T Saito¹, T Fukui¹, H Takeda¹, H Ohya³ and S Kawata¹

¹ Second Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan
² Yamagata Research Institute of Technology, Yamagata 990-2473, Japan
³ Institute for Life Support Technology, Yamagata Public Corporation for Development of Industry, Yamagata 990-2473, Japan

Correspondence to:
Correspondence to:
Dr H Togashi, Second Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan;htogashi{at}med.id.yamagata-u.ac.jp

Background: Sulfhydryl (SH) compounds are essential in maintaining mucosal integrity in the gastrointestinal tract. A decrease in colonic mucosal SH compounds affects the redox status of the mucosa, resulting in vulnerability to further attacks. Therefore, there is a strong need for in vivo evaluation of SH compounds in the colonic mucosa.

Aims: The aim of the current study was to establish a method of evaluating levels of SH compounds in the colonic mucosa of live animals before and after induction of colitis.

Methods: Murine experimental colitis was induced by instillation of trinitrobenzene sulphonic acid (TNBS) dissolved in 50% ethanol into the colon via the anus. For evaluation of mucosal SH compounds in the colon, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL), a stable nitroxide radical, was instilled into the colonic lumen of live mice and the spin clearance rate was measured by L-band electron spin resonance (ESR) spectroscopy.

Results: Morphological study showed that mucosal damage was severe one or two days after TNBS instillation. The colonic mucosa started to regenerate at four days, and looked normal at seven days, after induction of colitis. The spin clearance rate of carbamoyl-PROXYL decreased significantly at 0.5, 1, 2, and 4 days after induction of colitis compared with mice before TNBS instillation. Surprisingly, although the colonic mucosa looked normal seven days after TNBS administration, the spin clearance rate still remained significantly slow. The spin clearance rate returned to normal 14 days after induction of colitis. The change in in vivo spin clearance rate was consistent with the time dependent change in mucosal reduced glutathione, a major component of SH compounds.

Conclusion: The spin clearance rate obtained by L-band ESR spectroscopy in combination with carbamoyl-PROXYL can give an estimate of the level of colonic mucosal SH compounds in live animals and is useful for evaluating the mucosal defence system against oxidative stress.

Keywords: electron spin resonance; experimental colitis; nitroxide radical; sulfhydryl compounds; trinitrobenzene sulphonic acid

Abbreviations: carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; ESR, electron spin resonance; GSH, reduced glutathione; IBD, inflammatory bowel disease; NEM, N-ethylmaleimide; SH, sulfhydryl; TNBS, trinitrobenzene sulphonic acid; ROS, reactive oxygen species

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