1. The metabolic fate of two specifically deuterated analogues of valproic acid (VPA), [2-2H1]VPA and [3,3-2H2]VPA, was studied in the rat following i.p. injection. 2. A total of 11 urinary metabolites of each labelled substrate were detected by g.l.c.-mass spectrometry. Those metabolites which resulted from oxidation of the drug at C-4 and/or C-5 retained the deuterium label(s), whereas products of oxidation at C-2 and/or C-3 exhibited varying degrees of deuterium loss. 3. The deuterium content of 3-hydroxy-VPA indicated that this metabolite has a dual origin, and arises in part by beta-oxidation of VPA and in part by direct hydroxylation at C-3. An apparent intramolecular isotope effect (kH/kD) of ca. 8 was associated with the latter process. 3-Oxo-VPA appeared to be formed mainly by oxidation of delta 2-VPA, rather than by oxidation of 3-hydroxy-VPA. 4. Evidence was obtained that delta 3-VPA is formed reversibly from delta 2-VPA, and that further desaturation of delta 3-VPA gives rise to a metabolite believed to have a 2,3'-diene structure. 5. The stable isotope method employed in this investigation represents a powerful technique for studies on the origin of drug metabolites and for the elucidation of complex metabolic inter-relationships in vivo.

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The enantiomers of 2-n-propyl-4-pentenoic acid (delta 4-VPA), a known hepatotoxic metabolite of 2-n-propylpentanoic acid (valproix acid, VPA), were synthesized with the aid of the chiral auxiliaries (4S)-4-(2-propyl)-2-oxazolidone and (4R,5S)-4-methyl-5-phenyloxazolidone. Alkylation of the n-valeryl derivatives of these oxazolidones with allyl bromide, followed by reductive cleavage and chromic acid oxidation of the product, afforded the desired acids, (R)- and (S)-delta 4-VPA. Greater than 93% enantiomeric excess was achieved in the preparation of both enantiomers. Preliminary studies on the metabolic fate of (R)- and (S)-delta 4-VPA in freshly isolated rat hepatocytes revealed striking differences in the biotransformation of the two enantiomers. Quantification of two major metabolites of delta 4-VPA, viz., 4,5-diOH-VPA gamma-lactone and 2-n-propyl-2(E),4-pentadienoic acid (delta 2E,4-VPA), indicated that larger amounts of the gamma-lactone were formed in incubations utilizing (R)-delta 4-VPA as substrate, whereas production of the diene was greater in incubations with (S)-delta 4-VPA. On the basis of the premise that delta 4-VPA serves as a mechanism-based irreversible inhibitor of enzymes of the fatty acid beta-oxidation complex, these differences in metabolism suggest that the two enantiomers of delta 4-VPA may differ in their hepatotoxic potential.

The enantiomers of 2-[( 3-13C]-n-propyl)pentanoic acid [(R)- and (S)-[13C]VPA] were employed as metabolic probes to investigate stereochemical aspects of the biotransformation of valproic acid (VPA) to 2-n-propyl-4-pentenoic acid (delta 4-VPA), a hepatotoxic metabolite of VPA. When incubated with hepatocytes freshly isolated from untreated male rats, each labeled substrate (initial concentration 1.0 mM) underwent metabolism to [13C]-delta 4-VPA, the formation of which was time-dependent and occurred at a rate of ca. 20 ng/(10(6) cells.4-h incubation). Analysis of this unsaturated metabolite by GC-MS techniques revealed that, following incubation of (R)-[13C]VPA, desaturation had taken place preferentially (by a factor of approximately 4) on the labeled propyl group (i.e., on the R side chain). Parallel incubations with (S)-[13C]VPA supported this conclusion, in that metabolism of this isotopic variant of VPA led to a terminal olefin that also was predominantly (83 +/- 2%) of R configuration (in this case oxidized selectively on the unlabeled side chain). Hence, biotransformation of VPA to delta 4-VPA in rat hepatocytes occurs with marked enantiotopic differentiation, favoring production of the R enantiomer of this chiral metabolite. When rats were pretreated with phenobarbital (80 mg kg-1 day-1 ip for 3 days) prior to isolation of hepatocytes, the overall rate of metabolism of VPA to delta 4-VPA over the 4-h incubation period increased approximately 3-fold, while the degree of product enantioselectivity was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

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