HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A

Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products c...

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Bibliographic Details
Published in:Journal of lipid research 2002-07, Vol.43 (7), p.1133-1142
Main Authors: Chithalen, James V, Luu, Luong, Petkovich, Martin, Jones, Glenville
Format: Article
Language:English
Subjects:
Animals
Chromatography, High Pressure Liquid - methods
Cricetinae
Cricetulus
Cytochrome P-450 Enzyme System - genetics
Cytochrome P-450 Enzyme System - metabolism
cytochrome P450
HeLa Cells
Humans
Ketoconazole - pharmacology
Mass Spectrometry - methods
RAI-1
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Retinoic Acid 4-Hydroxylase
retinoids
RNA, Messenger - genetics
RNA, Messenger - metabolism
Tretinoin - chemistry
Tretinoin - metabolism
vitamin A metabolism
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Summary:Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products comprises 4-OH-all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid, and 18-OH-all-trans-retinoic acid, and more polar products, partially identified as dihydroxy and mono-oxo, mono-hydroxy derivatives. These more polar products are presumed to result from multiple hydroxylations on the beta-ionone ring. The inter-relationship of initial and polar metabolites was inferred from both gene-dose and time-course experiments. Both initial and secondary metabolic steps after 4-oxo-all-trans-retinoic acid are ketoconazole-sensitive, suggesting that steps in the production of water-soluble metabolites are cytochrome P450-dependent.
ISSN:0022-2275
DOI:10.1194/jlr.m100343-jlr200