title: Studies on the chemical reactivity of 2-phenylpropionic acid 1-O-acyl glucuronide and S-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.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143-0446, USA.
authors.forename: Chunze
authors.initials: C
authors.lastname: Li
authors.forename: Leslie Z
authors.initials: LZ
authors.lastname: Benet
authors.forename: Mark P
authors.initials: MP
authors.lastname: Grillo
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004952:Esters / Q000737:chemistry; D020719:Glucuronides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D013058:Mass Spectrometry; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000214:Acyl Coenzyme A; D004952:Esters; D020719:Glucuronides; D010666:Phenylpropionates; D011506:Proteins; C047011:acryloyl-coenzyme A; C013025:hydratropic acid; D005978:Glutathione