Abstract
3,5,5-Trimethylhexanoyl (TMH)-ferrocene has been used to produce iron loading in whole animals and in cultured hepatocytes. Iron loading produced by TMH-ferrocene is highly efficient and, of the compounds used to produce iron loading in experimental systems, most closely mimics the loading patterns observed in the human disease hemochromatosis. Previous work with TMH-ferrocene has shown that TMH-ferrocene is degraded in vivo because the iron is released from the ferrocene nucleus. Because TMH-ferrocene is highly lipophilic and stable chemically, we hypothesize that this molecule indeed could be degraded enzymatically. To measure the breakdown of TMH-ferrocene, iron release from the molecule was analyzed using a Ferrochem II analyzer, which uses constant potential coulometry to measure the amount of ionic iron within a biological sample. In this study, we show that TMH-ferrocene is degraded by a microsomal enzyme that requires NADPH and molecular oxygen. The TMH-ferrocenase activity is heat labile, requires a physiologic temperature, is induced by phenobarbital, and is inhibited by carbon monoxide and piperonyl butoxide but not by dicoumarol. The enzyme follows Michaelis–Menten kinetics and has aKm of 58.5 μM and aVmax of 57.5 nmol Fe released/mg protein/min. We conclude that TMH-ferrocene is degraded by a phenobarbital-inducible cytochrome P-450.
Footnotes
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Send reprint requests to: Dr. Harriet C. Isom, Department of Microbiology and Immunology, H107, Milton S. Hershey Medical Center, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: hisom{at}psu.edu
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This work was supported by research grants from the National Institutes of Health (CA23931 and DK54482) to H.C.I.
- Abbreviations used are::
- 20-MC
- 20-methylcholanthrene
- β-NF
- β-naphtholflavone
- BROD
- benzyloxyresorufin-O-debenzylase
- CO
- carbon monoxide
- EROD
- ethoxyresorufin-O-deethylase
- FeNTA
- ferric nitrilotriacetate
- MROD
- methoxyresorufin-O-demethylase
- PB
- phenobarbital
- PCN
- pregnenelone 16α-carbonitrile
- PROD
- pentoxyresorufin-O-depentylase
- S9 supernatants
- postmitochondrial supernatants
- TLC
- thin-layer chromatography
- TMH-ferrocene
- 3,5,5-trimethylhexanoyl-ferrocene
- TMHFase
- TMH-ferrocenase
- Received May 13, 1998.
- Accepted October 15, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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