Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
D. C. Beitz
R. L. Horst
The use of Fourier transform [superscript]1H NMR to characterize vitamin D[subscript]2 metabolites is described. High-resolution spectra were obtained on 5 [mu]g of material using a 300-MHz FT spectrometer. The [superscript]1H NMR spectra of a variety of both synthetic and naturally occurring vitamin D[subscript]2 metabolites resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D[subscript]2. The C-21 methyl group is now assigned to the doublet appearing at [delta]1.01, whereas the C-28 signal corresponds to the doublet at [delta]0.90. The [superscript]1H NMR spectrum was instrumental in establishing the identity of a new vitamin D[subscript]2 metabolite, 24,26-dihydroxyvitamin D[subscript]2. This unique physiological metabolite was isolated from the plasma of a vitamin D[subscript]2-toxic cow, but was also detected in rats supplemented with physiologic amounts of the vitamin. The formation of 24,26-dihydroxyvitamin D[subscript]2 represented a major pathway for further metabolism of 24-hydroxyvitamin D[subscript]2 in rats, exceeding the formation 24,25-dihydroxyvitamin D[subscript]2. Standard bioassays revealed that 24,26-dihydroxyvitamin D[subscript]2 possessed very little biological activity and likely represents a deactivation pathway for 24-hydroxyvitamin D[subscript]2;The effects of age on the intestinal and kidney 1,25-dihydroxyvitamin D[subscript]3 steroid receptor were studied in male Fischer 344 rats. There was a marked decrease in the number of intestinal receptors in old animals relative to young, but there was no discernible difference in the percentage of receptors binding to DNA-cellulose. Immunoblots only weakly identified intact intestinal receptor from older rats following DNA-cellulose chromatography. Similar amounts of young intestinal receptor were easily identified as a single band at 52 K in immunoblots. The presence of an increased endogenous protease activity in older animals was implicated. The specific binding of receptor in kidney cytosols was not different in the two age groups, but there was a significant decrease in the percentage of receptors binding to DNA-cellulose from older rats. Immunoblots of kidney preparations from either young or old rats following DNA-cellulose chromatography revealed two protein bands at 52 K and 50 K that corresponded to receptor. These results demonstrated for the first time that multiple forms of the vitamin D receptor exist in a mammalian system.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Nicholas Joseph Koszewski
Koszewski, Nicholas Joseph, "Identification of vitamin D2 metabolites and age-related changes in the 1,25-dihydroxyvitamin D3 steroid receptor in male Fischer 344 rats " (1988). Retrospective Theses and Dissertations. 8863.