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

Abstract

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.

LITAF Mediation of Increased TNF-α Secretion from Inflamed Colonic Lamina Propria Macrophages

“…Treated animals were administered TNBS ( BioChemika , 1 M in H 2 O) (1.5 mg in 100 µL of 50% Ethanol/water solution) by enema under isoflurane anesthesia. Colonic tissue was dissected at 24–72-hours after TNBS treatment at times associated with acute inflammation in prior studies [16], [17], [18]…”

A quantitative analysis of carbon-ion beam-induced reactive oxygen species and redox reactions

“…• . If in coexistence with a reduced form glutathione (GSH), the oxoammonium cation can then be reduced to hydroxylamine by receiving a hydrogen atom from glutathione, which is the main case in an in vivo situation, (15)(16)(17) or reduced to make a stable compound with glutathione, which is the main case in in vitro conditions. (18,19) The latter reaction is irreversible.…”

Imaging of reactive oxygen species generated in vivo

“…SH compounds, including GSH and cysteine, can reduce carbamoyl-PROXYL to corresponding hydroxylamine, resulting in the disappearance of the ESR signal of carbamoyl-PROXYL. When SH compounds in some organs are markedly lowered by oxidative stress, the reduction rate of carbamoyl-PROXYL generated by the reaction of hydroxylamine and ROS is significantly delayed (14). In the present study, we succeeded in in vivo imaging of a ROS-activated spin probe (carbamoyl-PROXYL) using a spin probe, ACP, that produces ESR-detectable nitroxide upon reaction with ROS within the living body before the occurrence of massive hepatic injury.…”