In vivo formation of the thiol conjugates of reactive metabolites of 4-ene VPA and its analog 4-pentenoic acid.

The terminal olefin metabolite of valproic acid (VPA), 4-ene VPA, is an analog of the experimental hepatotoxin 4-pentenoic acid and is believed to play a role in the hepatotoxicity of VPA. The formation of glutathione and N-acetylcysteine conjugates of the putative electrophilic metabolites of 4-ene VPA and 4-pentenoic acid was studied in vivo in the rat. Animals were treated intraperitoneally with the sodium salts of VPA, 4-ene VPA, (E)-2,4-diene VPA, 4-pentenoic or (E)-2,4-pentadienoic acids, and methylated bile and urine extracts were analyzed by LC/MS/MS and GC/MS techniques. The alpha,beta-unsaturated reactive metabolite of 4-pentenoic acid, 3-oxo-4-pentenoic acid, was isolated and identified as its thiol conjugates. In contrast, 3-oxo-4-ene VPA or its thiol conjugates could not be demonstrated as metabolites even though synthetic standards were available to facilitate their detection. Isolation of the thiol conjugates of 3-oxo-4-pentenoic acid provides the first direct spectroscopic evidence for the in vivo formation of the metabolite of 4-pentenoic acid considered responsible for the irreversible inhibition of fatty acid metabolism. The biliary and urinary metabolite profiles of 4-ene VPA and 4-pentenoic acid revealed basic differences between the in vivo metabolism of these two unsaturated carboxylic acids.