|Reinhardt, Timothy - Tim|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract only
Publication Acceptance Date: 12/2/1998
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: 1,25-Dihydroxyvitamin D3 (1,25-D3) undergoes side-chain cleavage leading to formation of C-23 acid. This metabolite is thought to be the terminal product in the deactivation of 1,25-D3. 1,25-D2 is also known to undergo side-chain oxidation. We studied the further metabolism of 1,25-D2 by using the C-ring labeled substrate [9,11-3H]-1,25-D2 (50-60 Ci/mmole). We utilized T47D 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 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-D strate. The reaction was allowed to occur over a period of 0-18 h with continuous shaking at 37 C. Termination was affected by the addition of 3.75 vol of MeOH:CHCl3 (2:1). Phase separation was achieved and lipid soluble metabolites were extracted by addition of CHCl3 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% alcohols in hex/MeCl2. The aqueous soluble metabolites were detected using an Econosphere**TM C18 HPLC column developed in the presence of 0.1% acetic acid using a gradient of 33-100% ACN in H2O. We established the presence of several lipid soluble metabolites. 1,24,25-D2, the major form, 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, peaking at 16-18 h. These data are the first to suggest the presence of aqueous soluble metabolites originating from 1,25-D2.