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Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit

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Summary

Cytochrome P-450 dependent oxygenase (3′-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3′-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, V max for 3′-hydroxy-hexobarbital formation and for hexobarbital-dependent H2O2 formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital tratment to 15.2 and 13.4 nmoles/mg/min. In phenobarbital treated rabbits, V max was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H2O2 formation. Spectral affinity constants (K s) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of hexobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H2O2 was higher than hydroxylation (V max:7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constants for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested.

It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe3+O2 2−), rendering either substrate-Fe3+O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.

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Abbreviations

S:

substrate

PB:

phenobarbital

HB:

hexobarbital

3-OH-HB:

3′-hydroxy-hexobarbital

DPH:

diphenylhydantoin

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Authors and Affiliations

  1. Institut für Klinische Pharmakologie, Freie Universität Berlin, Klinikum Steglitz, Hindenburgdamm 30, D-1000, Berlin 45

    Gerhard Heinemeyer, Santosh Nigam & Alfred G. Hildebrandt

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  1. Gerhard Heinemeyer
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  2. Santosh Nigam
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  3. Alfred G. Hildebrandt
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Supported in part by the Deutsche Forschungsgemeinschaft, Grant Hi 156/2, Biochemische Grundlagen der Arzneimittel- und Fremdstoffwirkungen

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Heinemeyer, G., Nigam, S. & Hildebrandt, A.G. Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit. Naunyn-Schmiedeberg's Arch. Pharmacol. 314, 201–210 (1980). https://doi.org/10.1007/BF00504539

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  • DOI: https://doi.org/10.1007/BF00504539

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