Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS)*1

Abstract

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 1α,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 1α-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 1α,25-(OH)2D3 at similar rates in vitro, but that for 25-OH-D3 but not 1α,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 1α,24,25-(OH)2D3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [3H]1α-OH-D2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.