|Reinhardt, Timothy - Tim|
Submitted to: Iowa State University Biochemistry Biophysics and Molecular Biology Symposi
Publication Type: Abstract Only
Publication Acceptance Date: 11/5/1999
Publication Date: N/A
Citation: N/A Interpretive Summary:
Technical Abstract: Calcitroic acid, a water soluble metabolite, is thought to be the terminal product of the deactivation of 1,25-dihydroxyvitamin D2 (1,25-(OH)2D2). 1,25(OH)2D2 is known to undergo side chain oxidation similar to 1,25- (OH)2D3. This study uses a novel 1,25-(OH)2D2 compound-tritiated on its C-ring at positions 9 and 11. HPK1a-ras cells were incubated with 1.4 mM cold and 0.6 mCi [9,11-3H]-1,25-(OH)2D2 for 96 h. Termination was achieved by harvesting the cells with trypsin-EDTA. Kidney perfusions were performed, incubating with 5 uM 1,25-(OH)2D2 for 8 h. Extraction was begun by adding 2 vol of methanol. Adjusting the pH to 8 allowed for the acidic metabolites to be retained in the aqueous layer while the lipid soluble metabolites were extracted twice with 1 vol of dichloromethane. The pH of the remaining aqueous phase was adjusted to 4.5 to protonate any acidic compounds and phase extracted twice with 1 vol of chloroform. Aqueous soluble metabolites were chromatographed on a Supelco**TM LC-18-DB column equipped with a guard column and eluted with a gradient of 39-100% acetonitrile in 0.1% acetic acid in water. The major aqueous metabolite comigrated with calcitroic acid. It also comigrated with calcitroic acid on a Supelco**TM Suplex pkb-100 column eluted isocratically with acetonitrile:water:acetic acid (60:40:0.1). The putative calcitroic acid from in vitro studies was methylated using an Aldrich MNNG-diazomethane kit and comigrated with methyl calcitroate on a Supelco**TM LC-18-DB column eluted with methanol:water (75:25). It also comigrated with methyl calcitroate on an Altech Econosphere silica column eluted with hexane:isopropanol (96:4). These data suggest that calcitroic acid is the terminal product of the deactivation of 1,25-dihydroxyvitamin D2.