Impairment of intestinal glutathione synthesis in patients with inflammatory bowel disease

Abstract

Background: Reactive oxygen species contribute to tissue injury in inflammatory bowel disease (IBD). The tripeptide glutathione (GSH) is the most important intracellular antioxidant.

Aims: To investigate constituent amino acid plasma levels and the GSH redox status in different compartments in IBD with emphasis on intestinal GSH synthesis in Crohn's disease.

Methods: Precursor amino acid levels were analysed in plasma and intestinal mucosa. Reduced (rGSH) and oxidised glutathione (GSSG) were determined enzymatically in peripheral blood mononuclear cells (PBMC), red blood cells (RBC), muscle, and in non-inflamed and inflamed ileum mucosa. Mucosal enzyme activity of gamma-glutamylcysteine synthetase (gamma GCS) and gamma-glutamyl transferase (gamma GT) was analysed. Blood of healthy subjects and normal mucosa from a bowel segment resected for tumor growth were used as controls.

Results: Abnormally low plasma cysteine and cystine levels were associated with inflammation in IBD (p < 10(-4)). Decreased rGSH levels were demonstrated in non-inflamed mucosa (p < 0.01) and inflamed mucosa (p = 10(-6)) in patients with IBD, while GSSG increased with inflammation (p = 0.007) compared with controls. Enzyme activity of gamma GCS was reduced in non-inflamed mucosa (p < 0.01) and, along with gamma GT, in inflamed mucosa (p < 10(-4)). The GSH content was unchanged in PBMC, RBC, and muscle.

Conclusions: Decreased activity of key enzymes involved in GSH synthesis accompanied by a decreased availability of cyst(e)ine for GSH synthesis contribute to mucosal GSH deficiency in IBD. As the impaired mucosal antioxidative capacity may further promote oxidative damage, GSH deficiency might be a target for therapeutic intervention in IBD.

Figure 1

Simplified diagram of mucosal glutathione synthesis in the ileum. GSH, glutathione; GSSG, glutathione disulphide; Gly, glycine; CysSH, cysteine; Cys-Cys, cystine; Glu, glutamate; AA, amino acid; γ-Glu-AA, γ-glutamyl-amino acid; γ-Glu-CysSH, γ-glutamylcysteine; Cys-Gly, cysteinylglycine. (1) Site of action of γ-glutamyl transferase; (2) site of action of γ-glutamylcysteine synthetase. For further information see introduction.

Figure 2

Fasting plasma cysteine levels in patients with Crohn's disease (n=33) and ulcerative colitis (n=33) before surgery and 10 days and three months after complete resection of inflamed bowel. Bars represent mean (SE). Data were statistically analysed by the Student's t test for unpaired samples including a Bonferroni's correction. *p<0.008, †p<10^-4 versus controls (n=65).

Figure 3

Linear regression analysis of preoperative plasma cysteine levels plotted against the individual clinical activity index according to Rachmilewitz in patients with ulcerative colitis.

Figure 4

Total thiol levels in non-deproteinised plasma in patients with Crohn's disease (n=33) and ulcerative colitis (n=33) before surgery and 10 days and three months after complete resection of inflamed bowel. Values represent means (SE) and were statistically analysed by the Student's t test for unpaired samples. According to Bonferroni's correction the level of significance was set at p<0.008. *p<10^-4 versus controls (n=65).

Figure 5

Pro-oxidant glutathione status in the mucosa of freshly resected ileum in patients with Crohn's disease. Reduced glutathione (rGSH) is decreased in inflamed (n=26) as well as non-inflamed (n=21) mucosa in Crohn's disease. Oxidised glutathione (GSSG) is increased in areas of inflammation so that the redox status of glutathione, as defined by the ratio rGSH/GSSG, is heavily decreased in inflamed mucosa. Bars represent mean (SE). Statistical analysis was performed by the Student's t test including a Bonferroni's correction. **p<0.01, †p<10^-4 versus controls (n=21).

Figure 6

Decreased mucosal enzyme activity of γ-glutamylcysteine synthetase (γGCS) in inflamed (n=26) and non-inflamed (n=21) ileum of patients with Crohn's disease. Bars represent mean (SE). Statistical analysis was performed by the Student's t test for unpaired samples including a Bonferroni's correction. *p<0.025, †p<10^-4 versus controls (n=21).

Figure 7

Decreased mucosal enzyme activity of γ-glutamyl transferase (γGT) in inflamed (n=26) and non-inflamed (n=21) ileum of patients with Crohn's disease. Bars represent mean (SE). Statistical analysis was performed by the Student's t test for unpaired samples. According to Bonferroni's correction the level of significance was set at p<0.025. *p<10^-4 versus controls (n=21).