Selective, rapid removal of the vitamin D-binding protein and its sterol ligands from human and bovine plasma.
Rapid and selective removal of plasma vitamin D-binding protein was effected by the serial passage of plasma over four columns of agarose containing covalently linked skeletal muscle G-actin. By maintaining an actin-to-binding protein molar ratio of at least 4 to 1 throughout, greater than 99% of the binding protein was removed from the fourth column's eluate. In contrast, 87% of the total plasma or serum protein applied was recovered, and electrophoretic analyses of human and bovine sera that had undergone these affinity chromatography steps revealed no major alterations in protein distribution. The procedure also removes vitamin D sterols selectively, with preference for 25-hydroxycalciferol (90% removal) over 1,25-dihydroxycalciferol (65-70% removal) and calciferol (70% removal), in accordance with the known affinity displayed by the binding protein for these sterol ligands. Recovery of other serum constituents (cortisol, proteins, peptide hormones, calcium and alkaline phosphatase) was excellent, further confirming the selectivity of the technique. Utilizing vitamin D-deficient serum, serum depleted of the vitamin D-binding protein was not distinguishable from control serum in supporting the growth of human fibroblasts in vitro. In comparison with other methods to remove serum-binding protein or sterols, the present technique is more selective and can be used for mammalian and avian sera. Material so prepared could prove useful for studies of the cellular access, metabolism, and effects of vitamin D sterols in vitro.
D000199:Actins; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D013261:Sterols / Q000097:blood* / Q000302:isolation & purification; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000302:isolation & purification
D000199:Actins; D008024:Ligands; D013261:Sterols; D014809:Vitamin D-Binding Protein