CHARACTERIZATION OF PROFLAVINE METABOLITES IN RAINBOW-TROUT

Proflavine (3,6-diaminoacridine) has potential for use as an anti-infe ctive in fish, and its metabolism by rainbow trout was therefore studi ed. Fourteen hours after intraarterial bolus administration of 10 mg/k g of proflavine, three metabolites were found in liver and bile, and o ne metabolite was found in plasma using reversed-phase HPLC with UV de tection at 262 nm. Treatment with hydrochloric acid converted the thre e metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with beta-glucuronidase and sacchari c acid 1,4-lactone, a specific beta-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination o f UV-VIS absorption and mass spectra, HPLC-purified metabolites were i solated from liver. Data from these experiments suggested that the pro flavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuro nosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. Whe n synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix -assisted, laser desorption ionization, time-of-flight MS. In addition , they coeluted on HPLC under different mobile phase conditions. Final ly, the in vitro incubation with liver subcellular preparations confir med this characterization and provided the evidence that APG can be fo rmed by glucuronidation of AP or acetylation of PG.