The Fallacy of Using Adrenochrome Reaction for Measurement of Reactive Oxygen Species Formed During Cytochrome P450-Mediated Metabolism of Xenobiotics
- Division of Drug Delivery and Disposition, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
- Dr. Dhiren R. Thakker, Division of Drug Delivery and Disposition, School of Pharmacy, Beard Hall CB 7360, Room 303B, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail: dhiren_thakker{at}unc.edu
Abstract
The adrenochrome reaction (oxidation of epinephrine to adrenochrome) has been widely employed as a standard assay for reactive oxygen species, produced under a variety of conditions, including those produced during cytochrome P450 (CYP)-mediated oxidation of substrates such as cyclosporine. However, it has been reported that epinephrine and adrenochrome can be metabolized by hepatic microsomes and that adrenochrome can also be metabolized by NADPH-CYP reductase. Thus, in the present report, we provide evidence that measurement of adrenochrome cannot be used as an index of reactive oxygen species generated during CYP-mediated metabolism of xenobiotics because adrenochrome and its precursor, epinephrine, interact with the CYP enzyme system as substrates and inhibitors. Our results indicated that adrenochrome was moderately stable in phosphate buffer but degraded rapidly (over 50% consumed in less than 2 min) by (cloned and expressed) CYP3A4 and CYP reductase in the presence of NADPH. Furthermore, both epinephrine and adrenochrome were found to be inhibitors of CYP3A4-mediated oxidation of testosterone. Together, these results lead to the conclusion that the use of adrenochrome reaction for measurement of reactive oxygen species formed during CYP3A4-mediated metabolism of xenobiotics is inappropriate.
Footnotes
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↵1 Current address: Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Eastern Point Rd., Groton, CT 06340.
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This work was supported by Grant 9705-ARG-0003 from the North Carolina Biotechnology Center, Research Triangle Park, NC.
- Abbreviations:
- CYP
- cytochrome P450
- CYP3A4OR
- cytochrome P450 3A4 and human cytochrome P450 reductase
- HPLC
- high-pressure liquid chromatography
- λmax
- absorbance maximum
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- Received July 25, 2001.
- Accepted October 16, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
