Characterization of halothane oxidation by hepatic microsomes and purified cytochromes P-450 using a gas chromatographic mass spectrometric assay

L. D. Gruenke, K. Konopka, Dennis Koop, L. A. Waskell

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Abstract

A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 μM in 100 μl of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes for phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90% in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.

Original languageEnglish (US)
Pages (from-to)454-459
Number of pages6
JournalJournal of Pharmacology and Experimental Therapeutics
Volume246
Issue number2
StatePublished - 1988
Externally publishedYes

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Halothane
Microsomes
Cytochrome P-450 Enzyme System
Gases
Trifluoroacetic Acid
Liver
Isoenzymes
Rabbits
Phenobarbital
Cytochromes b5
Proteins
Biotransformation
imidazole
Ethanol
Ions

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Characterization of halothane oxidation by hepatic microsomes and purified cytochromes P-450 using a gas chromatographic mass spectrometric assay",
abstract = "A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 μM in 100 μl of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes for phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90{\%} in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.",
author = "Gruenke, {L. D.} and K. Konopka and Dennis Koop and Waskell, {L. A.}",
year = "1988",
language = "English (US)",
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pages = "454--459",
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T1 - Characterization of halothane oxidation by hepatic microsomes and purified cytochromes P-450 using a gas chromatographic mass spectrometric assay

AU - Gruenke, L. D.

AU - Konopka, K.

AU - Koop, Dennis

AU - Waskell, L. A.

PY - 1988

Y1 - 1988

N2 - A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 μM in 100 μl of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes for phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90% in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.

AB - A sensitive assay for trifluoroacetic acid, the major product of the oxidative metabolism of halothane, has been developed to study the biotransformation of halothane. A selected ion monitoring gas chromatographic mass spectrometric assay measured trifluoroacetic acid levels as low as 1 μM in 100 μl of reaction mixture. This assay was used to quantitate halothane metabolism in human and rabbit microsomal systems and with purified proteins. Trifluoroacetic acid production was examined as a function of the concentration of substrate present, the amount of microsomal protein used and the length of reaction time. Halothane metabolism in microsomes was linear for at least 30 min, and up to a microsomal protein concentration of 1 mg/ml. In rabbits, phenobarbital and imidazole induced the microsomal metabolism of halothane 7.36- and 18.2-fold, respectively. Imidazole was used because it is a potent inducer of cytochrome P-450 isozyme 3a which is also induced by ethanol. The cytochrome P-450 in microsomes from a single human subject metabolized halothane at a rate comparable to that found in microsomes for phenobarbital- and imidazole-pretreated rabbits. The purified phenobarbital and imidazole inducible cytochromes P-450, isozymes 2 and 3a, catalyzed the oxidation of halothane to trifluoroacetic acid. Cytochrome b5 stimulated the isozyme 3a-catalyzed oxidation of halothane by 19-fold, whereas isozyme 2 catalyzed oxidation was increased 4.3-fold. Antibodies to cytochrome P-450 3a inhibited halothane metabolism by 90% in microsomes from imidazole-pretreated rabbits, suggesting that isozyme 3a catalyzes halothane metabolism in imidazole-pretreated rabbits. In conclusion, the oxidation of halothane to trifluoroacetic acid by cytochrome P-450 isozymes 3a and 2 is enhanced markedly by cytochrome b5.

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