STUDIES ON THE REACTIVITY OF ACYL GLUCURONIDES .8. GENERATION OF AN ANTISERUM FOR THE DETECTION OF DIFLUNISAL-MODIFIED PROTEINS IN DIFLUNISAL-DOSED RATS

Acyl glucuronide metabolites of carboxylic drugs such as the salicylat e derivative diflunisal (DF) have been shown to react with proteins to produce covalent adducts. To aid in the study of the formation and di stribution of these adducts in both humans and rats, we raised an anti serum against human serum albumin modified by covalent attachment of D F via an amide bond, using a carbodiimide reagent. This antiserum had wide reactivity, reacting with all types of DF-modified proteins teste d and with free DF (albeit at a lower affinity). It did not cross-reac t with other salicylates or other nonsteroidal anti-inflammatory drugs . The antiserum has been used in immunoblotting to detect proteins cov alently modified by DF in the plasma and livers of rats treated with t he drug for 7 days. Although some cross-reactivity was apparent on the blots, a series of DF-modified proteins was found in cytosolic, mitoc hondrial and mixed membrane fractions of hepatocytes, with molecular w eights ranging from 28 to 130 kDa.