Abstract
High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.
Highlights
From the Liver Study Unit, Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510
Glutathione Hydrolysis within the Biliary Tree-Having confirmed that glutamate and glycine are present in rat bile in high concentrationss, ubsequent experiments were designed to determine whether these amino acids in bile are derived from the hydrolysis of biliary glutathione
The enzyme y-glutamyl transferase is localized to the luminal surface of the membranes lining the biliary tract [4,5,6,7,8,9,10,11,23]
Summary
High concentrations of glutathione (GSH)and two of found that glutamate was the most abundant among a variety its constituent amino acids, glutamateand glycine, are of amino acids in rat bile, with a concentration between 0.67 normallyfound in rat bile. Prior to hydrolysis designed to determine whether the high concentrawithin the biliary tree, substantial concentrations of tions of glutamate and glycine in rat bile result from the GSH must be transported from liver cells into bile; hydrolysis of GSH, following the latter’s excretion into canminimal canalicular concentrations of this tripeptide alicular bile. DEM, 1mmol/kg in 2 ml of corn oil/kg, was given intraperitoneally in four animals after a 30-min control bile collection interval. GSSG eluted between glutamate and glycine, and did not interfere with the analysis of any of the amino acids reported in this study. Differences were considered to be statistically significant when p < 0.05
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