Abstract
Clofibric acid (p-chlorophenoxyisobutyric acid) is metabolized in vivo to a thioester-linked glutathione conjugate,S-(p-chlorophenoxyisobutyryl)glutathione (CA-SG). The formation of this metabolite is presumed to occur via transacylation reactions between glutathione (GSH) and reactive acyl-linked metabolite(s) of the drug. The present study examines the chemical reactivity of clofibryl-S-acyl-CoA (CA-SCoA), an acyl-CoA thioester intermediary metabolite of clofibric acid, with GSH to form the CA-SG in vitro. Incubations of CA-SCoA (1 mM) with GSH (5 mM) were carried out at pH 7.5 and 37°C, with analysis of the formed reaction products by isocratic reverse-phase high-performance liquid chromatography (HPLC). Results showed a time-dependent and linear formation of CA-SG up to 4 h (50 μM CA-SG formed/h), and after a 1-day incubation, the reaction mixture contained 0.7 mM CA-SG. The identity of CA-SG was confirmed by analysis of HPLC-purified material by tandem mass spectrometry. The rate of CA-SG formation was found to be increased 3-fold in incubations containing rat liver glutathione S-transferases (4 mg/ml). Analysis of the chemical stability of CA-SCoA in buffer at 37°C and varying pH showed the derivative to be stable under mildly acidic and basic aqueous conditions but to hydrolyze at pH values greater than 10 after a 1-day incubation (t1/2 = ∼1 day at pH 10.5). Results from these studies show that CA-SCoA is a reactive thioester derivative of clofibric acid and is able to acylate GSH and other thiol-containing nucleophiles in vitro and, therefore, may be able to acylate protein thiols in vivo, which could contribute to the toxic side effects of the drug.
Footnotes
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↵1 Present address: Pharmacia, Department of Drug Metabolism Research, 301 Henrietta St., Kalamazoo, MI 49007.
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This work was supported in part by National Institutes of Health Grant GM36633. Preliminary accounts of this work were presented at the American Association of Pharmaceutical Scientists Meeting, Seattle, WA, 1996.
- Abbreviations used are::
- CA
- clofibric acid
- CA-SG
- clofibryl-S-acyl-glutathione
- GSH
- glutathione
- CA-SCoA
- clofibryl-S-acyl-CoA
- 1-O-CAG
- 1-O-acyl-clofibryl glucuronide
- NAC
- N-acetylcysteine
- THF
- tetrahydrofuran
- LSIMS
- liquid secondary-ion mass spectrometry
- CID
- collisional-induced dissociation
- MS/MS
- tandem mass spectrometry
- HPLC
- high-performance liquid chromatography
- GST
- glutathione S-transferase
- DTT
- dithiothreitol
- ESI/MS
- electrospray mass spectrometry
- Received August 10, 2001.
- Accepted October 5, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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