title: HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A.
abstract: 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.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, ON, K7L 3N6, Canada.
authors.forename: James V
authors.initials: JV
authors.lastname: Chithalen
authors.forename: Luong
authors.initials: L
authors.lastname: Luu
authors.forename: Martin
authors.initials: M
authors.lastname: Petkovich
authors.forename: Glenville
authors.initials: G
authors.lastname: Jones
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000072516:Retinoic Acid 4-Hydroxylase; D014212:Tretinoin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D000072516:Retinoic Acid 4-Hydroxylase; D007654:Ketoconazole