Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.     

Studies on vitamin D3 metabolism. Discrete liver cytosolic binding proteins for vitamin D3 and 25-hydroxyvitamin D3

Two separate liver cytosolic proteins have been partially purified and identified by their selectivity for binding either [1α,2α(n)-³H]vitamin D₃ or 25-hydroxy [26(27)-methyl-³H]vitamin D₃. The chromatographic properties of the two proteins were not distinguishable by ion-exchange nor were they dependent upon the vitamin D₃ nutritional status of the birds. However, in molecular exclusion chromatography, the binding proteins can be successfully resolved into two discrete entities. Their binding properties suggest that they are not identical with plasma vitamin D₃ binding protein.     

High-pressure liquid chromatography: separation of the metabolites of vitamins D2 and D3 on small-particle silica columns.

The high-pressure liquid chromatographic separation of all of the known metabolites of vitamin D(2) and vitamin D(3) found in biological fluids has been achieved. This technique has been successfully applied to the analysis of vitamin D mixtures, purification of vitamin D metabolites, and identification of radioactive peaks. Some theoretical bases for the observed resolutions are suggested.     

Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Differences in the metabolism of vitamin D2 and vitamin D3 by subcellular fractions from rat liver

The 25-hydroxylation of vitamin D2 and vitamin D3 was studied in the mitochondrial fraction from rat liver and in a reconstituted system containing cytochrome P-450 from rat liver microsomes. The mitochondrial fraction catalyzed the 25-hydroxylation of vitamin D3 at least two times more effectively than the 25-hydroxylation of vitamin D2. Microsomal cytochrome P-450 catalyzed an efficient 25-hydroxylation of vitamin D3, but no 25-hydroxylation of vitamin D2 could be detected. The present results show a difference in the 25-hydroxylation of vitamin D2 and vitamin D3 in rat liver in vitro.