Many compounds which contain the carboxylic acid functionality are converted metabolically to their corresponding acyl glucuronides via UDP-glucuronosyl transferase. Studies performed in our laboratory have shown that acyl glucuronides of carboxylic acid non-steroidal antiinflammatory drugs (NSAIDs), used as model carboxylic acid compounds, are reactive and bind irreversibly to protein, in vivo and in vitro. The generation of covalently bound adducts may be responsible for the severe hypersensitivity reactions involved with the use of a number of carboxylic acid-containing compounds. The mechanisms by which these acyl glucuronides bind to proteins and cause hypersensitivity reactions remains poorly defined, although recent studies in our laboratory have provided an insight into underlying chemical events at the molecular level. Thus, in vitro studies were performed whereby the acyl glucuronide of tolmetin, a carboxylic acid containing NSAID which has been shown to cause hypersensitivity reactions in patients taking the drug, was reacted with human serum albumin (HSA) under physiological conditions. HSA-tolmetin adducts were reduced, carboxymethylated and digested with trypsin. Tolmetin-containing tryptic peptides were purified by HPLC and analyzed by liquid secondary ion mass spectrometry (LSIMS) on a Kratos MS50 mass spectrometer operated by the UCSF Mass Spectrometry Facility. From this analysis, the determination of specific modified HSA tryptic peptides and the chemical nature of the drug-peptide covalent adducts were possible. Further mass spectrometric analyses were performed on tolmetin acyl glucuronide modified tryptic peptides by tandem mass spectrometry (MS/MS) using a Kratos concept IIHH four-sector tandem mass spectrometer operated in the UCSFMass Spectrometry Facility by Fred Walls. From these MS/MS analyses the modified peptide amino acid sequences as well as the sites and types of covalent modification were established. Thus, from these mass spectrometric experiments, tolmetin acyl glucuronide was shown to bind to nucleophilic protein residues of HSA by two distinct mechanisms. One mechanism involved the transacylation of lysine amines and serine hydroxyl groups by nucleophilic displacement of the glucuronic acid to form amide and ester linkages between the protein and drug, respectively. The second mechanism, which accounted for most of the irreversible binding, occurs by a mechanism where lysine-amines react with the straight-chain aldehyde form of acyl migration isomers of the acyl glucuronide in a Schiff's base fashion. The resulting imine intermediate then rearranges to a more stable product, where the drug is bound to the protein by a glucuronic acid lysine linkage. Lysine 199 was found to be the residue modified to the greatest extent. The focus of ongoing and future work is directed toward determining the reactivity of acyl glucuronides with HSA in vivo, and to investigate which types of adducts are involved. The mass spectrometric methodologies developed for the analysis fo HSA-tolmetin adducts formed in vitro, which were obtained through a very productive collaboration with the UCSFMass Spectrometry Facility, will be applied to the analysis of acyl glucuronide modified HSA formed in vivo.
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