Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease

S. J. McKenzie, M. S. Baker*, G. D. Buffinton, W. F. Doe

*Corresponding author for this work

Research output: Contribution to journalArticle

298 Citations (Scopus)

Abstract

Evidence of in vivo oxidant-induced injury in inflammatory bowel disease (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol redox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific probe [14C]- iodoacetamide (IAM), there was decreased labeling of a number of proteins indicating oxidation of thiol groups in CEC from inflamed mucosa compared to paired normal mucosa, especially the loss of thiol labeling of a 37-kD protein which was almost completely lost. The loss of reduced protein thiol status for the 37-kD band was paralleled by loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essential reduced cysteine (Cys149) at the active site. The identity of the 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inflammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OCI >> ChT > H2O2 > NO (52±3, 250±17, 420±12, 779±120 μM oxidant) and OCl >> ChT > NO > H2O2 (89±17, 256±11, 407±105, 457±75 μM oxidant), respectively, for GAPDH enzyme activity. This study provides direct evidence of in vivo oxidant injury in CEC from inflamed mucosa of IBD patients. Oxidation and inhibition of essential protein function by inflammatory cells is a potential mechanism of tissue injury that may contribute to the pathogenesis of the disease and supports the exploration of compounds with antioxidant activity as new therapies for IBD.

Original languageEnglish
Pages (from-to)136-141
Number of pages6
JournalJournal of Clinical Investigation
Volume98
Issue number1
Publication statusPublished - 1 Jul 1996
Externally publishedYes

Keywords

  • glyceraldehydephosphate dehydrogenase (E.C.1.2.1.12)
  • inflammatory bowel disease
  • protein oxidation
  • protein thiol
  • reactive oxygen species

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