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Mechanistic studies of valproic acid hepatotoxicity : identification and characterization of thiol conjugates Kassahun, Kelem


The severe hepatotoxicity of the antiepileptic drug valproic acid (VPA) is believed to be mediated through chemically reactive metabolites. The monounsaturated metabolite 4-ene VPA is steatogenic in the rat, and in a similar fashion to the hepatotoxin 4-pentenoic acid, is thought to be oxidized by mitochondria to a highly reactive α,β-unsaturated ketone, 3-keto-4-ene VPA. The tripeptide thiol, glutathione (GSH), is known to react with a variety of electrophilic compounds that have the potential to interact with cellular macromolecules. The identification and structural characterization of GSH conjugates provides a means of identifying short-lived unstable electrophiles and thus an insight into the mechanisms of toxicity. This thesis describes the synthesis and characterization of thiol conjugates of reactive metabolites derived from the in vivo metabolism of VPA, 4-ene VPA, (E)-2,4-diene VPA, and 4-pentenoic acid. A negative ion chemical ionization gas chromatographic/mass spectrometric (NICI/GC/MS) method for the determination of VPA and 14 of its metabolites in a single chromatographic run was developed. A combination of pentafluorobenzyl and trimethylsilyl derivatization resulted in the [M-181]̄̄̄ ̄anion as the base peak for all the metabolites measured. When these ions were monitored sensitivities in the low picogram range were achieved. The VPA metabolite profile was determined in pediatric patients on VPA monotherapy and on combined therapy with either carbamazepine or clobazam. 4-Ene- and (E)-2,4-diene-VPA were found to be minor metabolites with serum levels below 1% that of VPA. In patients on combined therapy with carbamazepine, the ω and ω-l pathways of VPA metabolism were induced, while products of β-oxidation were significantly decreased. Polytherapy had no significant effect on the serum levels of 4-ene- or (E)-2,4-diene-VPA. Rats were dosed intraperitoneally with 100 mg/kg of the sodium salts of VPA, 4-ene-, (E)-2,4-diene-VPA, 4-pentenoic or (E)-2,4-pentadienoic acids. Methylated bile extracts were analyzed by high pressure liquid chromatography and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for GSH conjugates while urine samples were analyzed by GC/MS and LC/MS for N-acetylcysteine (NAC) conjugates and other metabolites. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene-VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The structures of these metabolites were confirmed by comparing GC/MS or LC/MS properties of the isolated metabolites to those of synthetic standards. The GSH and NAC conjugates of (E)-2,4-diene VPA were chemically synthesized and their structures established to be (E)-5-(glutathion-S-yl)-3-ene VPA and (E)-5-(N-acetylcystein-S-yl)-3-ene VPA by nuclear magnetic resonance spectroscopy and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo (about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr). In rats treated with 4-pentenoic and/or (E)-2,4-pentadienoic acids the following conjugates were identified and characterized by synthesis: GSH and cysteine conjugates of 3-oxo-4-pentenoic acid, GSH and NAC conjugates of (E)-2,4-pentadienoic acid, and the NAC conjugate of acrylic acid. The results thus provided the first direct biochemical evidence for the in vivo formation of the metabolite of 4-pentenoic acid considered responsible for the irreversible inhibition of fatty acid metabolism. The results also revealed basic differences between the mitochondrial metabolism of 4-ene VPA and 4-pentenoic acid. The 3-keto-4-ene VPA and its GSH and NAC conjugates were synthesized in order to facilitate the in vivo identification of these compounds following the administration of VPA, 4-ene-, or (E)-2,4-diene-VPA to rats. However, neither the 3-keto-4-ene VPA nor its thiol derivatives were evident in any of the treatments. The NAC conjugate of (E)-2,4-diene VPA was also found to be a metabolite of VPA in patients. The level of the conjugate appeared to be higher in two patients who recovered from VPA-induced liver toxicity. The characterization of GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug.

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