Reversal of the diabetogenic action of alloxan by sulfhydryl compounds.
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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.
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1. The time-course of cumene hydroperoxide-induced changes in lipid peroxidation, protein sulfhydryl groups and chemiluminescence intensity was determined in human erythrocytes. 2. Increase in lipid peroxidation was maximal within 60 min of incubation and was paralleled by a decrease in protein sulfhydryl groups and an increase in chemiluminescence formation. 3. A standard assay system was established to investigate the protective effects of antioxidants and scavenger compounds on cumene hydroperoxide-induced chemiluminescence formation. 4. Chain-breaking antioxidants (i.e. butylated hydroxytoluene) and sulfhydryl compounds (i.e. dithiothreitol) were able to suppress chemiluminescence formation. 5. Our results suggested that secondary free radicals, as well as sulfhydryl groups of proteins are involved in cumene hydroperoxide-induced chemiluminescence formation.
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