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Therapeutic modulation of the urea cycle in hyperammonemia induced by valproate : mechanisms and pharmacological protective strategies
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Valproate (VPA) is a drug with a broad-spectrum of efficacy for the treatment of epilepsy and psychiatric disturbances. Despite its uncontested pharmacological importance, VPA-associated hyperammonemic encephalopathy (VHE) is still a major concern. The consequences of a symptomatic hyperammonemia (HA) may be serious and dramatic. Despite the key role of glutamate dehydrogenase (GDH) on the regulation of ammonia metabolism in the liver, few studies in the literature address the effect of this antiepileptic drug or other drugs on this protein function. The present work aims at gaining new insights into the interaction of VPA and its major mitochondrial products, valproyl-CoA and propionyl-CoA on GDH, a crucial enzyme for the control of mitochondrial energy homeostasis. Therefore, kinetic studies with purified bovine GDH were performed on both directions of GDH-catalyzed reaction, using a spectrophotometric method. The effect of VPA and its CoA intermediates on the enzyme activity was investigated as function of different substrates (2-oxoglutarate, glutamate and ammonium) at various concentration ranges. The results obtained suggest that VPA and its metabolites may affect liver GDH activity on both directions, highlighting the central role of this reversible reaction on the hepatic regulation of nitrogen metabolism.
In addition, we investigated the rescuing effect of one pharmacological agent on VPA-induced inhibition of N-acetylglutamate synthase (NAGS), using freshly isolated rat liver mitochondria. The hypothesis was tested by monitoring citrulline production after modulation of the mitochondrial reactions of the urea cycle. To this aim we set-up a novel method, not previously available in our lab, for citrulline identification and quantification in biological samples, using tandem mass spectrometry. The present preclinical research work provide encouraging data on the potential benefit of the tested molecule to compensate the inhibitory effect of VPA on the urea cycle and eventually prevent the unwanted HA in vivo.
The cumulative effects of the anticonvulsant drug on NAGS and GDH activity may provide valuable information for understanding the mechanistic basis of its effect on ammoniagenesis. Overall, results presented in this work will account to define new therapeutic strategies to prevent or correct the VPA-associated HA, which remains, to our days, largely supportive.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013
Palavras-chave
Valproate Hyperammonemia Hepatotoxicity N-Acetylglutamate Synthase Glutamate Dehydrogenase N-Carglumic acid Teses de mestrado - 2013