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

• Main Authors: James V. Chithalen, Luong Luu, Martin Petkovich, Glenville Jones
• Format: Article
• Language: English
• Published: Elsevier 2002-07-01
• Series: Journal of Lipid Research
• Subjects: vitamin A metabolism; retinoids; cytochrome P450; RAI-1
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520314760

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 β-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.