BIOACTIVATION OF A TOXIC METABOLITE OF VALPROIC ACID, (E)-2-PROPYL-2,4-PENTADIENOIC ACID, VIA GLUCURONIDATION - LC MS/MS CHARACTERIZATION OF THE GSH-GLUCURONIDE DICONJUGATES/

The hepatotoxicity of the anticonvulsant drug valproic acid may be ass ociated with the formation of potentially reactive metabolites, one of which is (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene VPA). This report describes the characterization of new GSH-related conjugates o f this diene. Bile samples collected from male Sprague-Dawley rats dos ed ip with (E)-2,4-diene VPA (100 mg/kg) were analyzed by LC/MS/MS. In itial Q1 parent ion scanning indicated that the daughter ions m/z 162 and 123 could be derived from the ions at m/z 624 and 480, respectivel y. Subsequent collision-induced dissociation (CID) of these parent ion s revealed a common neutral loss of 176 Da which is diagnostic for glu curonides. A similar neutral loss of 176 Da was observed in daughter i on spectra of the biliary metabolites arising from [H-2(7)]-4-ene VPA dosed ip to rats, where the ion fragments containing the VPA portion w ere 7 amu higher than those derived from the unlabeled drug, CID of th e ion at m/z 624 also gave fragments characteristic for GSH conjugates such as the loss of glycine and glutamate moieties. Based on the MS d ata, the metabolites were assigned the diconjugate structures -(glutat hion-S-yl)-3-pentenoyl)-beta-D-glucuronide (5-GS-3-ene VPA-glucuronide I, MH(+), 624) and the corresponding 5-NAC-3-ene VPA-glucuronide (MH( +), 480), Further proof of structural identity was obtained from H-1 N MR of HPLC-purified metabolites, The amount of biliary 5-GS-3-ene VPA- glucuronide I was 7-fold greater than the corresponding 5-GS-3-ene VPA , the sum of the two metabolites accounting for 6.6% of the dose. Incu bation of 1-O-(2-propyl-2,4-pentadienoyl)-beta-D-glucuronide (2,4-dien e VPA-glucuronide) with GSH in the presence or absence of GST enzyme l ed to the formation of 5-GS-3-ene VPA-glucuronide I which was readily detected by LC/MS/MS, suggesting that in vivo the diconjugate may aris e from the reaction of GSH with 2,4-diene VPA-glucuronide. To our know ledge, this is the first recorded instance in which glucuronide format ion activates a drug to further conjugate with GSH via a Michael addit ion reaction.