title: Inhibition of 3-methylcrotonyl-CoA carboxylase explains the increased excretion of 3-hydroxyisovaleric acid in valproate-treated patients.
abstract: BACKGROUND
  Valproic acid (VPA) is a widely used anticonvulsant drug which affects mitochondrial metabolism including the catabolism of fatty acids and branched-chain amino acids.
  
  
  OBJECTIVE
  To elucidate the effect of valproate on the leucine pathway through a targeted metabolomics approach and the evaluation of the effects of valproate on the activity of biotinidase and 3-methylcrotonyl-CoA carboxylase (3MCC).
  
  
  METHODS
  Urine organic acid analysis was performed in patients under VPA therapy and healthy controls using gas-chromatography/mass spectrometry (GC-MS). Biotinidase activity was determined in plasma samples of both groups using an optimized spectrophotometric assay. After immunoprecipitation of short-chain enoyl-CoA hydratase (crotonase, ECHS1), 3MCC activity was measured in human liver homogenate using high-performance liquid chromatography (HPLC), in the absence and presence of valproyl-CoA.
  
  
  RESULTS
  The levels of 3-hydroxyisovaleric acid (3OH-IVA), one secondary metabolite of the leucine pathway, were significantly elevated in human urine after VPA treatment. Biotinidase activity in plasma samples ranged from very low to normal levels in treated patients as compared with controls. Enzyme activity measurements revealed inhibition of 3-methylcrotonyl-CoA carboxylase by valproyl-CoA (IC(50) = 1.36 mM). Furthermore, we show that after complete immunoprecipitation of crotonase in a human liver homogenate, 3-hydroxyisovaleryl-CoA is not formed.
  
  
  CONCLUSIONS
  Our results suggest the interference of VPA with the activity of 3MCC through a potential cumulative effect: direct inhibition of the enzyme activity by the drug metabolite valproyl-CoA and the inhibition of biotinidase by valproate and/or its metabolites. These interactions may be associated with the skin rash and hair loss which are side effects often reported in VPA-treated patients.
authors.affiliation: Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculdade de Farmácia da Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Paula B M
authors.initials: PB
authors.lastname: Luís
authors.forename: Jos P
authors.initials: JP
authors.lastname: Ruiter
authors.forename: Lodewijk
authors.initials: L
authors.lastname: IJlst
authors.forename: Luísa
authors.initials: L
authors.lastname: Diogo
authors.forename: Paula
authors.initials: P
authors.lastname: Garcia
authors.forename: Isabel Tavares
authors.initials: IT
authors.lastname: de Almeida
authors.forename: Marinus
authors.initials: M
authors.lastname: Duran
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Wanders
authors.forename: Margarida F B
authors.initials: MF
authors.lastname: Silva
mesh_terms: D043523:Biotinidase / Q000378:metabolism; D019735:Carbon-Carbon Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D055432:Metabolomics / Q000379:methods; D008956:Models, Chemical; D014631:Valerates / Q000378:metabolism*; D014635:Valproic Acid / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D014631:Valerates; C004961:beta-hydroxyisovaleric acid; D014635:Valproic Acid; D043523:Biotinidase; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase; D007930:Leucine