This chapter explains the use of chick kidney to enzymatically generate radiolabeled 1,25-dihydroxyvitamin D and other vitamin D metabolites. With the realization that vitamin D is metabolized to hydroxylated derivatives prior to performing its biologic function of calcium and phosphate regulation, it has become necessary to obtain these metabolites for biochemical, physiologic, and clinical studies. Of fundamental importance is the availability of radiolabeled active metabolites for use in competitive binding assays, receptor analyses, and in further metabolism studies. Because 25-(OH)[3H]D3 is available in very high specific activities (∼100 Ci/mmol), it was undertaken in this study to develop a reliable procedure for biosynthetically preparing 1,25-(OH)2[3H]D3 and 24,25-(OH)2[3H]D3 from this common precursor molecule. This technique involves the use of the renal lα-hydroxylase (lα-OHase) or 24-hydroxylase (24-24-OHase) enzyme systems obtained from chickens, and the isolation and purification of the desired product by high performance liquid chromatography (HPLC). This chapter describes this procedure after first detailing the properties of the renal lα-OHase enzyme of vitamin D-deficient chicks. This enzyme is of course pivotal in the biosynthesis, in vitro, of the most active lα-hydroxylated D-vitamins. The chapter also discusses the protocol for inducing the 24-OHase enzyme in chicks, and its utilization in the in vitro generation of 24-hydroxylated metabolites of vitamin D.
ASJC Scopus subject areas
- Molecular Biology