Complete β-oxidation of valproate: cleavage of 3-oxovalproyl-CoA by a mitochondrial 3-oxoacyl-CoA thiolase

Autor: Margarida F. B. SILVA, Jos P. N. RUITER, Henk OVERMARS, Albert H. BOOTSMA, Albert H. van GENNIP, Cornelis JAKOBS, Marinus DURAN, Isabel TAVARES de ALMEIDA, Ronald J. A. WANDERS
Rok vydání: 2002
Předmět:
Zdroj: Biochemical Journal. 362:755-760
ISSN: 1470-8728
0264-6021
DOI: 10.1042/bj3620755
Popis: The β-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with 3H-labelled VPA. The metabolism of [4,5-3H2]VPA and [2-3H]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Δ2(E)-valproyl-CoA,3-hydroxyvalproyl-CoA and 3-oxovalproyl-CoA (labelled and non-labelled) were determined using continuous on-line radiochemical and UV detection. The formation of these intermediates was investigated using the two tritiated precursors in respiratory states 3 and 4. Valproyl-CoA was present at highest concentrations under both conditions. Two distinct labelled peaks were found, which were identified as 3H2O and [4,5-3H2]3-oxo-VPA. The formation of 3H2O strongly suggested that VPA underwent complete β-oxidation and that [4,5-3H2]3-oxo-VPA was formed by hydrolysis of the corresponding thioester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mito chondrial lysate was incubated with synthetic 3-oxovalproyl-CoA, carnitine and carnitine acetyltransferase for subsequent monitoring of the formation of propionylcarnitine and pentanoylcarnitine using electrospray ionization tandem MS. The detection of these compounds demonstrated unequivocally that the intermediate 3-oxovalproyl-CoA is a substrate of a mitochondrial thiolase, producing propionyl-CoA and pentanoyl-CoA, thus demonstrating the complete β-oxidation of VPA in the mitochondrion. Our data should lead to a re-evaluation of the generally accepted concept that the biotransformation of VPA by mitochondrial β-oxidation is incomplete.
Databáze: OpenAIRE
Externí odkaz: