Abstract
Oxidative conversion of all-trans-retinol (t-ROH) to all-trans-retinal (t-RAL) is recognized as the rate-limiting step for biosynthesis of all-trans-retinoic acid fromt-ROH in mammalian hepatic tissues. The purpose of this study was to investigate the role of human cytochrome P-450 (CYP)-dependent monooxygenation in the conversion oft-ROH to t-RAL. Adult human liver microsomes (HLMS) were incubated with t-ROH, and retinoids generated were identified and quantified by liquid chromatography-mass spectroscopy, HPLC, and other methods. HLMS-catalyzed generation of t-RAL fromt-ROH was primarily NADPH-dependent and was strongly inhibited by carbon monoxide. Rates of reactions increased linearly with time and concentrations of HLMS, and exhibited classical substrate saturation. These observations strongly indicated that the reaction proceeded via CYP-catalyzed monooxygenation. On the basis of responses to selective chemical inhibitors, isoforms from CYP family 1 and the CYP3A subfamily appeared to be very active. Members of the CYP2C subfamily and CYP2D6 exhibited lesser activities and CYP2A6, CYP2B6, and CYP2E1 were virtually inactive. cDNA-expressed human CYP enzymes (CYP SUPERSOMES) also were used to assess the capacity of individual CYP enzymes to catalyze the reaction. Based on responses to selective chemical inhibitors, specific activities, and levels present in adult human hepatic tissues, CYP1A2 and CYP3A4 strongly appeared to be the major CYP enzymes catalyzing hepatic oxidative conversion oft-ROH to t-RAL in the adult human liver. CYP1A1 and CYP1B1 SUPERSOMES both exhibited exceptionally high activities, and in extrahepatic tissues, these isoforms could play important roles in biosynthesis of all-trans-retinoic acid from t-ROH.
Footnotes
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Send reprint requests to: M. R. Juchau, Ph.D., Department of Pharmacology, School of Medicine, Box 357280, University of Washington, Seattle, WA 98185. E-mail: juchau{at}u.washington.edu
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This work was supported by National Institute of Environmental Health Sciences Grant ES-04041.
- Abbreviations used are::
- t-RA
- all-trans-retinoic acid
- t-ROH
- all-trans-retinol
- t-RAL
- all-trans-retinal
- HLMS
- human liver microsomes
- CYP
- cytochrome P-450
- ANF
- α-naphthoflavone
- TAO
- troleandomycin
- LC-MS
- liquid chromatography-mass spectroscopy
- CO
- carbon monoxide
- Received July 20, 1999.
- Accepted November 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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