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 studies focused on: (1) elucidating the role of CYP24 in 25-OH-D3 and 1alpha,25-(OH)2D3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D3 metabolism in CYP24-knockout (CYP24-XO) cells and; (4) comparing 1alpha-OH-D2 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-D3 and 1alpha,25-(OH)2D3 at similar rates in vitro, but that for 25-OH-D3 but not 1alpha,25-(OH)2D3, this rate is strongly influenced by the concentration of DBP. Unlike their wild type littermates, the administration of 25-OH-D3 to CYP24-XO mice results in no measurable 24,25-(OH)2D3 production. When neonatal murine keratinocytes are prepared from wild type and CYP24-XO mice there was no measurable production of 24,25-(OH)2D3 or 1alpha,24,25-(OH)2D3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [3H] 1alpha-OH-D2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.
Masuda
Sonoko
S
Department of Biochemistry, Queen's University, Kingston, Ont., Canada. masudas@post.queensu.ca
Kaufmann
Martin
M
Byford
Valarie
V
Gao
Mian
M
St-Arnaud
René
R
Arabian
Alice
A
Makin
Hugh L J
HL
Knutson
Joyce C
JC
Strugnell
Stephen
S
Jones
Glenville
G
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D014807:Vitamin D / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase