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 the transport of vitamin D and of 25-hydroxyvitamin D in human plasma.

A study was conducted to investigate whether human plasma contains one or more than one protein for the transport of vitamin D and of 25-hydroxyvitamin D (25-OH-D).Serum was labeled in vivo with a mixture of radioactive vitamin D3 (derived from orally administered tracer vitamin D3) and of endogenously synthesized labeled 25-OH-D3. Samples of such serum were subjected to several different protein fractionation procedures. Only a single peak of protein-bound radioactivity was observed after each of these procedures. The fraction comprising the ascending and the descending limbs of the single peak of protein-bound radioactivity (after each procedure) were separately pooled. In each instance the ratio of radioactive 25-OH-D3 to radioactive vitamin D3 was found to be almost identical in both the ascending and the descending limbs. Taken together, these findings provide strong evidence that human serum contains only a single binding protein responsible for the normal transport of both vitamin D and 25-OH-D. Plasma labeled in vitro with added 3H-labeled 25-OH-D3 was subjected to gel filtration on Sephadex G-200 and to chromatography on columns of DEAE-cellulose and of SP-Sephadex. After each of these procedures a single peak of protein-bound radioactivity was observed, with elution profiles of protein and of radioactivity that were identical with those observed with in vivo labeled serum. These data indicate that tracer 25-OH-D3 added to plasma in vitro binds to the same plasma protein normally responsible for the transport of vitamin D and of 25-OH-D.     

Regulation of vitamin D metabolism.

Vitamin D can be regarded as a prohormone and its most potent metabolite, 1, 25-dihydroxyvitamin D₃, a hormone which mobilizes calcium and phosphate from bone and intestine. In true hormonal fashion, the biosynthesis of 1, 25-dihydroxyvitamin D₃ by kidney mitochondria is feed-back regulated by serum calcium and serum phosphorus levels. The lack of calcium brings about a secretion of parathyroid hormone which stimulates 1, 25-dihydroxyvitamin D₃ synthesis while low blood phosphorus stimulates 1, 25-dihydroxyvitamin D₃ synthesis even in the absence of the parathyroid glands. For such regulation to occur, vitamin D must be present probably because 1, 25-dihydroxyvitamin D₃ itself is needed for the regulation. The molecular and cellular mechanisms whereby 1, 25-dihydroxyvitamin D₃ synthesis is regulated are unknown despite many recent reports. Likely the elucidation of these mechanisms must await a detailed investigation of the enzymology of the renal 25-hydroxyvitamin D₃-1α-hydroxylase. In addition to the regulation at the 25-hydroxyvitamin D₃-1α-hydroxylase step, vitamin D metabolism is regulated at the hepatic vitamin D-25-hydroxylase level. This regulation is a suppression of the hydroxylase by the hepatic level of 25-hydroxyvitamin D₃ itself by an unknown mechanism. Much remains to be learned concerning the regulation of this newly discovered endocrine system but already the findings are not only relevant to calcium homeostasis but also to an understanding of a variety of metabolic bone diseases.     

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.     

The photobiogenesis and metabolism of vitamin D.

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. P450-dependent mixed function oxidase system is 1alpha,25(OH)2D. When calcium and phosphate homeostasis prevails the renal 25-OH-D-1alpha-hydroxylase activity is limited and instead a non-cytochrome P450 mixed function oxidase metabolizes 25-OH-D to 24R,25(OH)2D. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitary hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined. The regulation of the photometabolism of provitamin D3 to vitamin D3, the role and significance of the side-chain metabolism of 1,25(OH)2D by the small intestine, and the metabolism of 25-OH-D to 24R,25(OH)2D by chondrocytes and its stimulation of protein synthesis in these cells are just a few issues that will require further investigation.     

Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.

We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.     

Ligand recognition by the vitamin D receptor.

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on, is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1,25-(OH)(2)D(3) (OCT), 20-epi-1,25-(OH)(2)D(3) and 20-epi-22-oxa-24,26,27-trihomo-1,25-(OH)(2)D(3). In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1,25-(OH)(2)D(3) and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands.     

Vitamin D supplements and 25-hydroxy vitamin D concentrations in the elderly.

Serial 25-hydroxy vitamin D (25-OHD) concentrations were measured in long-stay geriatric patients treated with vitamin D. Comparison between a treatment and a control group showed that a daily dose of 500 IU vitamin D produced a significant increase in 25-OHD levels by two months. The supplement had a striking effect when the initial 25-OHD level was low and very little effect when it was high. 25-OHD levels in subjects on 2000 IU vitamin D daily were only marginally higher than those in subjects on 500 IU. A dose of 500 IU vitamin D daily should therefore produce adequate blood 25-OHD concentrations in most old people, and probably prevent most cases of osteomalacia in the elderly--though a large-scale study is needed to confirm this.     

Three-dimensional structure-function relationship of vitamin D and vitamin D receptor model

On the basis of conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we have suggested the active space region concept of vitamin D: The vitamin D side-chain region was grouped into four regions (A, G, EA and EG) and the A and EA regions were suggested to be important for vitamin D actions. We extended our theory to known highly potent vitamin D analogs and found a new region F. The analogs which occupy the F region have such modifications as 22-oxa, 22-ene, 16-ene and 18-nor. Altogether, the following relationship between the space region and activity was found: Affinity for vitamin D receptor (VDR), EA > A> F > G > EG; Affinity for vitamin D binding protein (DBP), A > G,EA,EG; Target gene transactivation, EA > F > A > EG > or = G; Cell differentiation, EA > F > A > EG > or = G; Bone calcium mobilization, EA > GA > F > or = EG; Intestinal calcium absorption, EA = A > or = G > EG. We modeled the 3D structure of VDR-LBD (ligand binding domain) using hRARgamma as a template, to develop our structure-function theory into a theory involving VDR. 1alpha,25(OH)(2)D(3) was docked into the ligand binding pocket of the VDR with the side chain heading the wide cavity at the H-11 site, the A-ring toward the narrow beta-turn site, and the beta-face of the CD ring facing H3. Amino acid residues forming hydrogen bonds with the 1alpha- and 25-OH groups were specified: S237 and R274 forming a pincer type hydrogen-bond for the 1alpha-OH and H397 for the 25-OH. Mutants of several amino acid residues that are hydrogen-bond candidates were prepared and their biologic properties were evaluated. All of our mutation results together with known mutation data support our VDR model docked with the natural ligand.     

Serum concentrations of 25-hydroxy vitamin D in Europeans and Asians after oral vitamin D3.

Serum concentrations of 25-hydroxy vitamin D (25-OHD3) were measured in seven Asians of Indian extraction and eight Europeans before and at intervals after taking 1 mg vitamin D3 by mouth. In all subjects the concentrations rose in the 24 hours after ingestion. There was little change over the next nine days in the concentrations in the Europeans but those in the Asians continued to rise until about day 10. Subsequent rates of fall in 25-OHD3 were similar in the two groups. Our observations suggest that the low serum concentrations of 25-OHD3 found in Asians are not caused by either impaired intestinal absorption of vitamin D or rapid clearance of 25-ODH3 from the plasma.     

Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata.

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T(4). The G-T(4) levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T(4) was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T(4) was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T(4) on the uptake of Ca(45) was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca(45) increased in T(4) treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca(45) into axes was not affected by exogenous T(4) until day 10 of the study. These data suggest that G-T(4) may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more 'irregular' spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

Rapid actions of vitamin D compounds.

The rapid actions of vitamin D compounds are surveyed in a variety of target tissues, including intestine, muscle, bone, hepatocytes, fibroblasts, HL-60 cells, kidney, mammary gland, and parathyroid. Evidence for non-nuclear receptors vs. membranophilic effects is discussed, followed by a consideration of signal transduction mechanisms including steroid hormone activated Ca2+ channels, phospholipid metabolism, protein kinases, and the role of G-proteins.