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

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 stu...

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Bibliographic Details
Published in:Journal of steroid biochemistry and molecular biology 2004-05, Vol.89 (1-5), p.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: Article
Language:English
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
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Summary: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.
ISSN:0960-0760
1879-1220
DOI:10.1016/j.jsbmb.2004.03.094