Complete beta-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 (3)H-labelled VPA. The metabolism of [4,5-(3)H(2)]VPA and [2-(3)H]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 (3)H(2)O and [4,5-(3)H(2)]3-oxo-VPA. The formation of (3)H(2)O strongly suggested that VPA underwent complete beta-oxidation and that [4,5-(3)H(2)]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 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.
Silva
Margarida F B
MF
Department of Clinical Chemistry and Paediatrics, University of Amsterdam, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
Ruiter
Jos P N
JP
Overmars
Henk
H
Bootsma
Albert H
AH
van Gennip
Albert H
AH
Jakobs
Cornelis
C
Duran
Marinus
M
Tavares de Almeida
Isabel
I
Wanders
Ronald J A
RJ
D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002331:Carnitine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017666:Deuterium Oxide / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008930:Mitochondria, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D014635:Valproic Acid / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*
C452022:3-oxovalproyl-CoA; D000214:Acyl Coenzyme A; D014635:Valproic Acid; D000102:Acetyl-CoA C-Acyltransferase; D017666:Deuterium Oxide; D002331:Carnitine