|Reinhardt, Timothy - Tim|
Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: 10/1/1999
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D30 is known to be deactivated to calcitroic acid (C23 acid) by a series of oxidation reactions at C24 and C23. Like 1,25-(OH)2D3, 1,25-(OH)2D2 is also known to undergo side chain oxidation; however, there has been no evidence presented suggesting that 1,25-(OH)2D2 undergoes side chain cleavage. To investigate this possibility, we used HPK1a-ras-cells incubated with 1.4 uM 1,25-(OH)2D2, along with [9,11-3H]-1,25-(OH)2D2 (0.6 uCi/ml media). We used the isolated rat kidney model perfused with 5 uM 1,25-(OH)2D2. Cells were harvested at various time points up to 96 h. Perfusate from the kidneys was harvested after 8 h. Extraction of the 1,25-(OH)2D2 metabolites from the cell and kidney preparations was initiated by the addition of 2 vol of methanol. The pH of the extracts were adjusted to 8.0 and extracted twice with 1 vol of dichloromethane. Aqueous soluble metabolites were extracted twice with chloroform at pH 4.5. Aqueous soluble metabolites were subjected to reverse phase HPLC analysis. The major aqueous soluble metabolite from both the kidney and cell incubations was putatively identified as C23 acid since it comigrated with authentic C23 acid on 2 reverse phase HPLC columns. The putative C23 acid from the cell incubations was methylated and found to comigrate with methylated authentic standard on straight phase and reverse phase HPLC columns. Identification of the methylated metabolite was confirmed by mass spectral analysis. The putative C23 acid from the kidney perfusions and intermediates leading to the formation of C23 acid in the 1,25-(OH)2D2 pathway are currently being studied. These data suggest that C23 acid is a major terminal product of the deactivation of 1,25-(OH)2D2.