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

The beta-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with H-3-labelled VPA. The metabolism of [4,5-H-3(2)]VPA and [2-H-3]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Delta(2(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 (H2O)-H-3 and [4,5-H-3(2)]3-oxo-VPA. The formation of (H2O)-H-3 strongly suggested that VPA underwent complete beta-oxidation and that [4,5-H-3(2)]3-oxo-VPA was formed by hydrolysis of the corresponding thio-ester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mitochondrial 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 beta-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 beta-oxidation is incomplete.