P450-Catalyzed In-Chain Desaturation of Valproic Acid: Isoform Selectivity and Mechanism of Formation of Δ3-Valproic Acid Generated by Baculovirus- Expressed CYP3A1

The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Δ3-valproic acid (Δ3-VPA) by rat liver microsomes was examined. Microsomal rates of formation of Δ3-VPA were below quantifiable limits in reactions catalyzed by control female rat liver microsomes, but were induced more than 20-fold following pretreatment with triacetyloleandomycin and pregnenolone-16α-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2-2H1]VPA demonstrated that Δ3-VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Δ2-VPA. CYP3A1 expression was optimized in the baculovirus expression vector system, and infected insect cell membranes which were supplemented with P450 reductase catalyzed formation of 3-OH-, 4-OH-, 5-OH-, Δ3-, and Δ4-VPA in ratios of 160:35:6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3-2H2]VPA or [4,4-2H2]VPA, yieldedkH/kDvalues for Δ3-VPA of 2.00 ± 0.06 and 2.36 ± 0.08, respectively. These values were significantly lower than the isotope effects observed in the same incubations for 3-OH-VPA formation from 3,3-D2-VPA (kH/kD= 6.04 ± 0.08), or for 4-OH- and Δ4-VPA formation from 4,4-D2-VPA (kH/kD> 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochromeb5-linked CoA desaturases and suggest further that CYP3A1-dependent formation of Δ3-VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position.