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

Chem Res Toxicol. 1996 Mar;9(2):517-26. doi: 10.1021/tx950120y.


The hepatotoxicity of the anticonvulsant drug valproic acid may be associated 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 of this diene. Bile samples collected from male Sprague-Dawley rats dosed ip with (E)-2,4-diene VPA (100 mg/kg) were analyzed by LC/MS/MS. Initial Q1 parent in scanning indicated that the daughter ions m/z 162 and 123 could be derived from the ions at m/z 624 and 480, respectively. Subsequent collision-induced dissociation (CID) of these parent ions revealed a common neutral loss of 176 Da which is diagnostic for glucuronides. A similar neutral loss of 176 Da was observed in daughter ion spectra of the biliary metabolites arising from [2H7]-4-ene VPA dosed ip to rats, where the ion fragments containing the VPA portion were 7 amu higher than those derived from the unlabeled drug. CID of the ion at m/z 624 also gave fragments characteristics for GSH conjugates such as the loss of glycine and glutamate moieties. Based on the MS data, the metabolites were assigned the diconjugate structures 1-O-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-beta-D-glucur onide (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 1H NMR 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. Incubation of 1-O-(2-propyl-2,4-pentadienoyl)-beta-D-glucuronide (2,4-diene VPA-glucuronide) with GSH in the presence or absence of GST enzyme led 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 arise from the reaction of GSH with 2,4-diene VPA-glucuronide. To our knowledge, this is the first recorded instance in which glucuronide formation activates a drug to further conjugate with GSH via a Michael addition reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biliary Tract / chemistry
  • Biotransformation
  • Fatty Acids, Unsaturated / toxicity*
  • Glucuronates / analysis*
  • Glucuronates / metabolism*
  • Glucuronidase / biosynthesis*
  • Glutathione / analysis*
  • Glutathione / metabolism*
  • Male
  • Mass Spectrometry
  • Oxytocin / metabolism
  • Protein Binding
  • Rats
  • Rats, Sprague-Dawley
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Valproic Acid / metabolism*
  • Valproic Acid / toxicity*


  • Fatty Acids, Unsaturated
  • Glucuronates
  • Oxytocin
  • Valproic Acid
  • 2-propyl-2,4-pentadienoic acid
  • Glucuronidase
  • Glutathione