Studies on the Chemical Reactivity of 2-Phenylpropionic Acid 1-O-Acyl Glucuronide andS-Acyl-CoA Thioester Metabolites

Abstract

Chemically reactive species formed from the metabolism of carboxylic acid-containing compounds have been proposed as mediators of their toxic side-effects. Two alternative metabolic pathways known to be involved in the generation of reactive acylating metabolites of carboxylic acids are acyl glucuronidation and acyl-CoA formation. Here, we present studies with 2-phenylpropionic acid focused on evaluating the relative abilities of acyl glucuronides versus acyl-CoA derivatives to transacylate the nucleophilic cysteinyl-thiol of glutathione. Thus, synthetic 2-phenylpropionyl-S-acyl-CoA (2-PPA-SCoA) and biosynthetic 2-phenylpropionyl-1-O-acyl glucuronide (2-PPA-1-O-G) were incubated separately, and at varying concentrations (15.6-500 nM as well as at 0.1 mM), with GSH (1, 5, and 10 mM) in buffer (pH 7.4, 37 degrees C), and formation of the transacylation product, 2-phenylpropionyl-S-acyl-glutathione (2-PPA-SG), was quantified by reverse-phase HPLC and LC-MS. HPLC analysis of the products from both the reaction of 2-PPA-SCoA and 2-PPA-1-O-G with GSH showed the presence of 2-PPA-SG, which was confirmed by coelution with authentic 2-PPA-SG as well as by its LC/MS mass spectrum. The formation of 2-PPA-SG was time- and concentration-dependent with a formation rate constant of (1.9 +/- 0.2) x 10(-2) M(-1) x s(-1) from reactions of GSH with 2-PPA-SCoA, and (2.7 +/- 0.4) x 10(-4) M(-1) x s(-1) from reactions of GSH with 2-PPA-1-O-G. Therefore, the reactivity of 2-PPA-SCoA with GSH was 70 times greater than the reactivity of GSH with 2-PPA-1-O-G, which was found to acyl-migrate to less reactive isomers. Analysis of the in vitro stability of 2-PPA-SCoA and 2-PPA-1-O-G in the absence of GSH showed the CoA esters to be completely stable after 24 h, whereas the acyl glucuronides decomposed by 50% in 1.3 and 2.4 h of incubation at pH 7.4 and 37 degrees C for (R)- and (S)-2-PPA-1-O-G, respectively. In addition, studies of the reactivity of 2-PPA-SCoA with bovine serum albumin showed time- and pH-dependent covalent binding to the protein in vitro. These results support the hypothesis that xenobiotic acyl-CoA thioesters are reactive acylating species that, in addition to acyl glucuronides, may contribute to xenobiotic acid-protein adduct formation in vivo.

Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats

“…2. This fragmentation pattern is similar to that described for other acyl-CoAs Li et al, 2002;Olsen et al, 2003b;Sidenius et al, 2004), including that of the structurally related nonsteroidal anti-inflammatory drug, zomepirac (Olsen et al, 2005). The retention time and MS/MS spectrum of a synthetic standard were similar to those seen in liver samples from rats that had received a dose of 100 mg/kg.…”

“…An acyl glucuronide is one example of a reactive carboxylic acid metabolite (Smith et al, 1986), but recent studies on acyl-CoAs and acyl-glutathione conjugates have shown that thioester metabolites react more rapidly with thiols (glutathione/N-acetylcysteine) than the corresponding ester glucuronides Li et al, 2002;Olsen et al, 2002;Grillo et al, 2003).…”

“…Acyl-CoAs are intermediates in amino acid conjugation, carnitine conjugation, fatty acid synthesis, and ␤-oxidation. In vitro characterization of the chemical reactivity has shown that the acyl-CoAs are significantly more reactive than the corresponding acyl glucuronides in the spontaneous reaction with glutathione Li et al, 2002;Olsen et al, 2002). Therefore, covalent binding of xenobiotic carboxylic acids in vivo may occur via at least two mechanisms, the importance of which depends on the concentration and the relative reactivity of each reactive metabolite.…”

The Nonenzymatic Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid toward Amino and Thiol Functional Group Bionucleophiles

“…The enzyme-bound activated intermediate is then displaced by coenzyme A to yield the associated acyl-CoA product and free AMP (Vlahcevic et al, 1999). Acyl-CoA and acyl-AMP metabolites both spontaneously and enzymatically, via glutathione-Stransferase, form GSH-conjugates (Li et al, 2002;Grillo et al, 2012;Horng and Benet, 2013). In addition to GSH, glycine (Keller, 1842) and, taurine (James et al, 1971;Hutson and Casida, 1978) are two of the most commonly cited metabolic amino acid conjugation reactions.…”

Feasibility of Biomonitoring of Exposure to Permethrin Through Analysis of Long-Lived (Metabolite) Adducts to Proteins

“…On the basis of previous research , it is likely that the cysteine-34 residue will react readily with the activated glucuronides, but whether the resulting thioester adduct is stable enough to accumulate in the organism, is not very plausible. However, see also Grillo and Hua, 2003 for corresponding adducts of NSAID zomepirac) found that the S-glutathione adduct of the NSAID diclofenac was excreted from the bile of rats after administration of diclofenac glucuronide (see also Li et al, 2002). Protein binding of glucuronides of benzoic acids that are structurally related to 3-PBA has been reported (see, e.g., Akira et al, 2002).…”