RT Journal Article
SR Electronic
T1 Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry.
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 369
OP 376
VO 23
IS 3
A1 A Ding
A1 P Zia-Amirhosseini
A1 A F McDonagh
A1 A L Burlingame
A1 L Z Benet
YR 1995
UL http://dmd.aspetjournals.org/content/23/3/369.abstract
AB The structures of adducts formed from in vitro incubation of a drug (tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.