title: pH-dependent rearrangement of the biosynthetic ester glucuronide of valproic acid to beta-glucuronidase-resistant forms.
abstract: A major metabolite of the antiepileptic drug valproic acid (VPA) in animals and man is the glucuronic acid conjugate, which is cleaved by incubation with beta-glucuronidase (specific for 1-O-substituted-beta-D-glucopyranosiduronic acids) or hydrolysis in strong acid or alkali. Previous studies revealed that an often substantial proportion of the alkali-labile conjugated VPA in stored urine or bile samples was not hydrolyzed by beta-glucuronidase, suggesting the presence of nonglucuronide conjugates. In the present study, bile from a NaVPA-treated rat was preincubated at 37 degrees C for 3 hr at pH values from -0.8 to 12.9, and then analyzed for nonconjugated VPA, VPA released by hydrolysis with beta-glucuronidase, and VPA released by hydrolysis with alkali. At pH 3-7, all alkali-labile conjugated VPA remained susceptible to beta-glucuronidase hydrolysis, whereas at pH 0-3 and 7-11, a proportion became resistant to the enzyme. GLC and GC/MS analysis of trimethylsilyl derivatives of the intact conjugates revealed the appearance of seven additional peaks, adjacent to the biosynthetic ester glucuronide, of which six were structural isomers and one was a dehydrated species. The data were consistent with acid- and base-catalyzed intramolecular acyl migration of the valproate moiety away from the C-1 position, with subsequent processes of ring-opening, mutarotation, and lactonization yielding structural isomers and lactones which were not substrates for beta-glucuronidase. It was further shown that these rearrangements are time- and temperature-dependent. Consequently, sample handling and storage conditions of ester glucuronides prior to analysis are of prime importance, since hydrolysis with beta-glucuronidase is frequently used for identification and quantification of glucuronides.
mesh_terms: D000818:Animals; D001646:Bile / Q000378:metabolism*; D001711:Biotransformation; D002849:Chromatography, Gas; D008401:Gas Chromatography-Mass Spectrometry; D005966:Glucuronidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014635:Valproic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: C041285:valproic acid glucuronide; D014635:Valproic Acid; D005966:Glucuronidase