|Reinhardt, Timothy - Tim|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: 12/1/1998
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: 1,25-Dihydroxyvitamin D (1,25-D3) undergoes side-chain cleavage leading to the formation of the aqueous soluble C-23 acid. This metabolite is thought to be the terminal product in the deactivation of 1,25-D3. We studied the metabolism of 1,25-D2 by using the C-ring labeled substrate [9,11-3H]-1,25D2. We utilized T-47D cells which had been pretreated with 1.8 nM 1,25-D3 in order to stimulate the expression of the 24-hydroxylase. After 18 h exposure to the 1,25-D3, the cells were harvested and suspended in incubation buffer at a density of 10**7 cells/ml. Cells were aliquoted into 0.5 ml fractions and the reaction was initiated by the addition of 3 uCi of the [9,11-3H]-1,25-D2 substrate. The reaction was allowed to occur over 0-18 h with continuous shaking at 37 C. Termination was affected by the addition of 3.75 vol of methanol:chloroform (2:1). Phase separation was achieved and lipid soluble metabolites were extracted by the addition of chloroform and H2O. Aqueous soluble metabolites were those remaining following the removal of the lipid soluble material. The lipid soluble material was subjected to HPLC using a Zorbax**TM Sil column developed with a gradient of 3.8-8.0% alcohols in hexane/methylene chloride. The aqueous soluble metabolites were detected using a reverse phase Econosphere**TM C18 HPLC column developed in the presence of 0.1% acetic acid using a gradient of 33-100% acetonitrile in H2O. We established the presence of several lipid soluble metabolites with 1,24,25-D2 being the major form. 1,24,25-D2 peaked at 6-8 h and disappeared by 18 h. Corresponding to the decline of 1,24,25-D2 was the appearance of an aqueous soluble metabolite which peaked by 16-18 h. This aqueous soluble metabolite was distinct from either C-23 acid or C-22 acid on HPLC.