Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS)

• Published in: Journal of steroid biochemistry and molecular biology Vol. 89; no. 1-5; pp. 149 - 153
• Main Authors: Masuda, Sonoko, Kaufmann, Martin, Byford, Valarie, Gao, Mian, St-Arnaud, René, Arabian, Alice, Makin, Hugh L.J, Knutson, Joyce C, Strugnell, Stephen, Jones, Glenville
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Oxford Elsevier Ltd 01.05.2004; Elsevier Science
• Subjects: 1α-Hydroxyvitamin D 2; 24-Hydroxylation; Animals; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; CYP24; CYP24-knockout mouse; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; DBP; Fundamental and applied biological sciences. Psychology; LC/MS; Mass Spectrometry - methods; Mice; Mice, Knockout; Steroid Hydroxylases - genetics; Steroid Hydroxylases - metabolism; Vertebrates: endocrinology; Vitamin D - metabolism; Vitamin D catabolism; Vitamin D3 24-Hydroxylase; LC/MS; Vitamin D catabolism; DBP; 1α-Hydroxyvitamin D 2; CYP24; 24-Hydroxylation; Cytochrome P450; CYP24-knockout mouse; Rodentia; Liquid chromatography; CYP24-knockout mouse: Vitamin D catabolism; Metabolism; la-Hydroxyvitamin D2; Vertebrata; Mammalia; Mouse; Vitamin D; Animal; Catabolism; Mutation; Mass spectrometry
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2004.03.094

The development of novel gene expression systems for cytochrome P450s (CYPs) together with a revolution in analytical mass spectrometry with the emergence of liquid chromatography/mass spectrometry (LC/MS) has opened the door to answering some long-standing questions in Vitamin D metabolism. Our studies focused on: (1) elucidating the role of CYP24 in 25-OH-D 3 and 1α,25-(OH) 2D 3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D 3 metabolism in CYP24–knockout (CYP24–XO) cells and; (4) comparing 1α-OH-D 2 metabolism in the CYP24-XO mouse in vivo and in vitro. Methodology employed CYP24 over-expression and knockout systems in conjunction with state-of-the-art analytical LC/MS, diode array, and radioisotopic detection methods. We found that CYP24 metabolizes 25-OH-D 3 and 1α,25-(OH) 2D 3 at similar rates in vitro, but that for 25-OH-D 3 but not 1α,25-(OH) 2D 3, this rate is strongly influenced by the concentration of DBP. Unlike their wild type littermates, the administration of 25-OH-D 3 to CYP24-XO mice results in no measurable 24,25-(OH) 2D 3 production. When neonatal murine keratinocytes are prepared from wild type and CYP24-XO mice there was no measurable production of 24,25-(OH) 2D 3 or 1α,24,25-(OH) 2D 3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [ 3 H] 1α-OH-D 2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.