Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro.

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 degrees 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 microM 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 degrees 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 (t(1/2) = approximately 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.