beta-1-O- (NAG) and 2-O-glucuronides (2-isomer) of (S)-naproxen (NA) were prepared to determine which positional isomer(s) of the acyl glucuronide of NA is responsible for forming covalent adducts with human serum albumin (HSA). Their comparative stability and covalent binding adduct formation with HSA were investigated at pH 7.4 and at 37 degreesC. NA and its acyl glucuronides were simultaneously determined by HPLC. Three positional isomers were formed successively after incubation of NAG in the buffer only. However, when NAG was incubated with HSA (30 mg/mL), isomers other than the 2-isomer were formed in little or negligible quantities. In HSA solution, NAG (kd = 2.08 +/- 0.08 h-1) was four times less stable than 2-isomer (kd = 0.51 +/- 0.02 h-1). NAG was degraded by hydrolysis (khyd = 1.01 +/- 0.10 h-1) and isomerization (kiso = 1.07 +/- 0.07 h-1) to the same extent; however, hydrolysis was predominant for the 2-isomer (kd = 0.51 +/- 0.02 h-1). The incubation of both NAG and 2-isomer with HSA led to the formation of a covalent adduct; however, the adduct formation from the 2-isomer proceeded more slowly than that from NAG. The present results suggest that the covalent binding of NA to HSA via its acyl glucuronides proceeds through both transacylation (direct nucleophilic displacement) and glycation mechanisms; NAG rapidly forms an adduct that may be unstable, and the protein adduct from the 2-O-acyl glucuronide is as important for the covalent binding as those from the 1-O-acyl glucuronides.