Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction.     

Enumeration of denitrifying microbial populations in turf

Summary Denitrifer populations of a silt and silt loam soil under a Kentucky bluegrass turf were enumerated using the most probable number (MPN) procedure. The influence of soil texture, soil depth, soil moisture, and additions of nitrate fertilizer on denitrifier populations were determined. Saturated soil conditions increased denitrifier populations 87-fold in the silt soil and 121-fold in the silt loam soil. Denitrifier populations did not differ significantly between soil depths and additions of fertilizer nitrate did not influence populations.     

Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.