Stereochemistry of naturally-occurring 25-hydroxyvitamin D3-26,23 lactone as determined by radioligand binding analysis and high-performance liquid chromatography |
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Authors: | R L Horst T A Reinhardt D H Williams |
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Affiliation: | 1. National Animal Disease Center, Agricultural Research, U. S. Department of Agriculture, P. O. Box 70, Ames, Iowa 50010 USA;2. University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, England |
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Abstract: | The four stereoisomers of 25-hydroxyvitamin D3-26,23 lactone (25-OHD3-26,23 lactone) were tested against in vivo 25-OHD3-26,23 lactone to determine their relative competition in the radioligand binding assays for 25-OHD3 and 1,25-(OH)2D3. The 25R-OHD3-26,23S lactone and in vivo 25-OHD3-26,23 lactone behaved identically in the radioligand binding assay for 25-OHD3 and were ~5-fold more potent than 25-OHD3 at displacing 25-OH[3H]D3. The 25S-OHD3-26,23S lactone was the poorest competitor in this assay, requiring a 10-fold excess relative to 25-OHD3 to displace 50% of the 25-OH[3H]D3. The order of competition in the 25-OHD3 radioligand binding assay was 25R-OHD3-26,23S lactone = in vivo 25-OHD3-26,23 lactone ? 25S-OHD3-26,23R lactone > 25-OHD3 ? 25R-OHD3-26,23R lactone > 25S-OHD3-26,23S lactone. The order of competition in the 1,25-(OH)2D3 cytosol receptor assay was essentially reversed from the competition in the 25-OHD3 assay and was 25S-OHD3-26,23S lactone > 25-OHD3 ? 25S-OHD3-26,23R lactone > 25R-OHD3-26,23S lactone = in vivo 25-OHD3-26,23 lactone. When tested in a high-performance liquid chromatographic system which separates all four stereoisomers, the in vivo 25-OHD3-26,23 lactone comigrated with synthetic 25R-OHD3-26,23S lactone. These data firmly establish that the naturally-occurring 25-OHD3-26,23 lactone has the 25R, 23S stereochemistry. In addition, these data are the first to demonstrate that the four stereoisomers of 25-OHD3-26,23 lactone have different affinities for the plasma vitamin D binding protein and the 1,25-(OH)2D cytosol receptor. |
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Keywords: | 23 25-dihydroxy-vitamin D3 |
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