Identification of the mixed disulfide of glutathione and cysteinylglycine in bile: Dependence on γ-glutamyl transferase and responsiveness to oxidative stress

C. Madhu, Z. Gregus, C. C. Cheng, C. D. Klaassen

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Biliary excretion of glutathione disulfide (GSSG) is used as an index of oxidative stress. Analysis of endogenous thiols and disulfides in rat bile by reverse phase high performance liquid chromatography with electrochemical detection revealed an unknown disulfide which eluted immediately after GSSG. This disulfide was tentatively identified as the mixed disulfide of glutathione (GSH) and cysteinylglycine (Cys-Gly), based on its coelution on a reverse phase column with the synthetic GS-Cys-Gly. GS-Cys-Gly was also detected in bile of other species. On analyzing species differences in biliary excretion of GSH-related thiols and disulfides, it was concluded that biliary excretion of GS-Cys-Gly was related to the excretion of both GSSG and Cys-Gly, which is formed from GSH by γ-glutamyltransferase (γ-GT)-catalyzed hydrolysis. Species with low hepatic γ-GT (i.e., hamsters and mice) excreted little Cys-Gly in bile. These animals excreted negligible amounts of GS-Cys- Gly even when biliary excretion of GSSG was markedly increased by paraquat- induced oxidative stress. Rats and guinea pigs, which have high hepatic γ-GT activities, excreted large amounts of both Cys-Gly and GS-Cys-Gly. Treatment of rats with acivicin, an inhibitor of γ-GT, decreased the biliary excretion of both Cys-Gly and GS-Cys-Gly. Paraquat treatment of rats resulted in an increase in GSSG excretion with concomitant increase of GS-Cys-Gly excretion. Rabbits, which also have high hepatic γ-GT activity, excreted little GS-Cys- Gly into bile. This may be due to the fact that the hepatobiliary transport of GSH and GSSG is very low in rabbits. However, when the biliary excretion of GSSG was increased by paraquat administration, rabbits excreted large amounts of GS-Cys-Gly, even exceeding that of GSSG. These observations suggest that biliary GS-Cys-Gly originates from a thiol-disulfide exchange reaction between GSSG and Cys-Gly, with the latter being formed by γ-GT- catalyzed hydrolysis of GSH. Our results with paraquat confirm that increased biliary excretion of GSSG is a good indicator of oxidative stress in mice and hamsters (i.e., in species with low activity of hepatic γ-GT). However, in species with high hepatic γ-GT (i.e., rats and rabbits), the biliary excretion of GS-Cys-Gly may be supplemental or even superior to the biliary excretion of GSSG to indicate oxidative stress in vivo.

Original languageEnglish
Pages (from-to)896-900
Number of pages5
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
Publication statusPublished - Jan 1 1992


ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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