IDENTIFICATION OF BETA-GLUCURONIDASE-RESISTANT DIASTEREOMERIC GLUCURONIDES OF 3-HYDROXY-3-METHYLOXINDOLE FORMED DURING 3-METHYLINDOLE METABOLISM IN GOATS

Goats were jugularly infused with the pneumotoxin 3-methylindole (3MI; 15 mg/kg, 0.5 mu Ci/kg) dissolved in cremophor-EL to characterize the urinary metabolites of 3MI in a ruminant specie. Urine was collected for 36 hr after the beginning of a 2-hr infusion period, and 3MI metab olites were purified using reversed-phase HPLC. Goats excreted 3MI as at least 11 distinct metabolites, Metabolites were characterized using a combination of UV spectroscopy, H-1 and C-13-NMR spectroscopy, and negative-ion FAB/MS, Two of the metabolites (E(1) and E(2)) representi ng similar to-30% of the urinary radioactivity, were unambiguously ide ntified as diastereomeric glucuronides of 3-hydroxy-3-methyloxindole [ HMOI; 3-(beta-D-glucosiduronic acid)-3-methyloxindole]. Glucuronide co njugates were investigated using enzymatic and chemical hydrolysis. Th ese ethe-real glucuronides were unique in that they were not readily h ydrolyzable with bovine beta-glucuronidase, although one of the diaste reomers was hydrolyzed sparingly by beta-glucuronidase from Helix poma tia. Treatment of the glucuronides with 6 M HCl for a 2-hr period libe rated unconjugated HMOI, Treatment of each diastereomer with dilute ac id (pH 3) or dilute alkali (pH 10) was ineffective at hydrolyzing the conjugates. Goats form HMOI from 3MI and extensively glucuronidate the metabolite before excreting it, as opposed to mice that do not conjug ate HMOI before excretion, These ethereal glucuronic acid conjugates s eem to be unique in that they are essentially resistant to beta-glucur onidase-catalyzed hydrolysis.