title: Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.
abstract: Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl-cysteine products rather than the S-acyl-NAC conjugate.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0446, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Grillo
authors.forename: L Z
authors.initials: LZ
authors.lastname: Benet
mesh_terms: D000215:Acylation; D000818:Animals; D000924:Anticholesteremic Agents / Q000737:chemistry* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D002995:Clofibric Acid / Q000737:chemistry* / Q000493:pharmacokinetics; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry*; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
chemical_list: D000924:Anticholesteremic Agents; C438124:clofibryl-acyl-glutathione; D002995:Clofibric Acid; D005723:gamma-Glutamyltransferase; D005978:Glutathione