Helen L. Henry

Regulation of vitamin D metabolism

Fundamental to understanding the way in which perturbations in the vitamin D endocrine system can affect human health is an appreciation of the steps involved in the production of the well-recognized active hormonal form, 1,25-dihydroxyvitamin D(3). Thus this paper focuses first on the nature and regulation of the two enzymes responsible for the production of 1,25-dihydroxyvitamin D(3), the 25-hydroxylase in the liver and the 1α-hydroxylase in the kidney. The most important regulators of the 1α-hydroxylase in the kidney are 1,25-dihydroxyvitamin D(3) itself, parathyroid hormone and FGF23. The extent and importance of extra-renal, 1,25-dihydroxyvitamin D(3) synthesis is then considered. Finally the features of the 24R-hydroxylase, which produces 24R,25-dihydroxyvitamin D(3) in the kidney and is induced by and inactivated, 1,25-dihydroxyvitamin D(3)in target cells are described.

Inhibition of Parathyroid Hormone Secretion by 25-Hydroxycholecalciferol and 24,25-Dihydroxycholecalciferol in the Dog

We studied the effects of vitamin D metabolites on parathyroid hormone (PTH) secretion. Test materials were injected into the cranial thyroid artery of the dog, and immunoreactive PTH was measured frequently in serum samples from the inferior thyroid vein and the femoral vein. This model for the study of secretion had previously been validated with the use of known modulators on PTH secretion. In control experiments, injection of 100% ethanol, the vehicle in which cholecalciferol (D(3)) metabolites were suspended, resulted in no change in PTH secretion. Likewise, native vitamin D(3), in doses ranging from 250 to 1,250 ng had no effect on PTH secretion. 25-Hydroxycholecalciferol, 25-(OH)D(3), in doses of 125-240 ng, caused complete suppression of PTH secretion. When 24,25-dihydroxycholecalciferol, 24,25-(OH)(2)D(3), was injected in doses of 50-250 ng, suppression of PTH secretion was again complete; in doses of 5 ng, injection of this metabolite resulted in significant but incomplete suppression of secretion. In doses of 50-250 ng, 1,25-(OH)(2)D(3) strongly stimulated PTH secretion, but in a dose of 5 ng this metabolite had no effects. Injection of equal doses of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) resulted in significant suppression of PTH secretion. Hypocalcemia-induced stimulation of PTH secretion was suppressed by 24,25-(OH)(2)D(3) while hypercalcemia-induced suppression of PTH secretion was stimulated by 1,25-(OH)(2)D(3). In all experiments showing suppression of PTH secretion, peripheral PTH decreased. Arguments are presented for considering the suppressive effects of D(3) metabolites as physiologic modulators. However, this stimulating effect of 1,25-(OH)(2)D(3) occurred only in pharmacologic doses and hence probably has no physiologic relevance.

Studies on the mechanism of action of calciferol VII. Localization of 1,25-dihydroxy-vitamin D3 in chick parathyroid glands

When 1,25(OH)2-vitamin D3 was administered to vitamin D-deficient chicks, within two hours the parathyroid glands were observed to accumulate this steroid to a concentration four times that present in the blood and equivalent to levels observed in the target intestine. Similarly, when 25-(OH)-vitamin D3 was administered, the parathyroid glands had 2.4 times the concentration of the metabolite, 1,25-(OH)2-vitamin D3 as that seen in the blood and 60% of that found in the intestine. These results are consistent with the concept that the hormonally active form of vitamin D, 1,25-(OH)2-vitamin D3, may interact with the parathyroid glands to effect changes in parathyroid hormone secretion.

14th Vitamin D Workshop consensus on vitamin D nutritional guidelines.

As background information, the reader should appreciate that at he 13th Vitamin DWorkshop in 2006 it was agreed that about half f elderly North Americans and Western Europeans and probably lso two thirds of the rest of the world are vitamin D deficient as udgedby their inability tomaintain a healthy bonedensity [1–3]. It as also generally agreed that the serum concentration of 25(OH)D n normal subjects is the best indicator for judging the vitamin D tatus in patients with vitamin D-related disease states [4]. The14thWorkshoponVitaminD,held inBrugge, Belgium,Octoer 4–8, 2009wasattendedby419 scientists from35countrieswho ere privileged to listen and participate in a Vitamin D Roundtable hat was held in order to allow presentation and broad discusion of two distinct views of and approaches to worldwide vitamin nutritional status. One Roundtable position is that an absolute inimum 25(OH)D level of 20ng/ml (50nmol/l) is necessary in all ndividuals in order to support and maintain all the classic actions f vitamin D on bone andmineral health and that, according to this riterion, a large proportion of the world’s population is vitamin D eficient. Thosewhohold this position further believe that thehuge ffort needed to ameliorate this deficiency must be undertaken as oon and actively as possible and that the target 25(OH)D levels of 20ng/ml should be obtained in the majority of the target populaion. The second Roundtable position is that newer data showing ssociations between vitamin D status and prevalence of several iseases such as cardiovascular disease, hypertension, colon and reast cancer, multiple sclerosis as well as the involvement of vitain D in muscle strength and immune functions [5], indicates that arget levels of 25(OH)D should be 30–40ng/ml (75–100nmol/l) t the minimum. As a basis for policy decision making, these two ositions are incompatible with one another. However, through onsideration of the aspects of vitamin D nutrition upon which the roponents of the two views agree, as well as acknowledging diferences in opinion, consensus on how to proceed in the near term an emerge. This was the goal of the Vitamin D Roundtable. TheRoundtable, chaired byAnthonyNorman (USA) andChristoher Gallagher (USA), began with 15-min presentations from obert Heaney (USA) and Reinhold Vieth (Canada), both propoents of 25(OH)D>40ng/ml, followed by presentations by Roger ouillon (Belgium) and Paul Lips (Netherlands),who advocateminmum25(OH)D levels of 20ng/ml. A selected group of experts from round theworld, Bess Dawson-Hughes (USA), John Pettifor (South frica), Peter Ebeling (Australia), and Christine Lamberg-Allardt

Studies on the mode of action of calciferol: Biological activity of 1α,24R,25-trihydroxyvitamin D3 in the chick☆

Abstract The biological activity of 1α,24 R ,25-trihydroxyvitamin D 3 [1α,24 R ,25(OH) 3 D 3 ] was elevated in comparison to the hormonally active form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], in the rachitic chick in terms of its ability to (a) stimulate intestinal calcium absorption, (b) mobilize bone calcium, (c) induce intestinal calcium binding protein, (d) modulate the level of enzyme activity of the renal 25-OH-D 3 -1-hydroxylase system, and (e) interact with the intestinal cystosol-chromatin receptor system for the 1α,25(OH) 2 D 3 receptor system. In each of these assays, the relative ratio of activity of 1α,24 R ,25(OH) 3 D 3 to 1α,25(OH) 2 D 3 was (a) 25–50, (b) ca. 20, (c) 10, (d) 50, and (e) 36%, respectively.

Different shapes of the steroid hormone 1α,25(OH)2-vitamin D3 act as agonists for two different receptors in the vitamin D endocrine system to mediate genomic and rapid responses☆

Vitamin D(3) produces biologic responses as a consequence of its metabolism into 1alpha,25(OH)(2)-vitamin D(3) [1alpha,25(OH)(2)D(3)] and 24R,25(OH)(2)-vitamin D(3). The metabolic production of these two seco steroids and their generation of the plethora of biologic actions that are attributable to the parent vitamin D(3) are orchestrated via the integrated operation of the vitamin D endocrine system. This system is very similar in its organization to that of classic endocrine systems and is characterized by an endocrine gland (the kidney, the source of the two steroid hormones), target cells which possess receptors for the steroid hormones, and a feed-back loop involving changes in serum Ca(2+) that alter the secretion of parathyroid hormone (a stimulator of the renal 1-hydroxylase) which modulates the output by the kidney of the steroid hormones. There are, however, at least two unique aspects to the vitamin D endocrine system. (a) The chemical structures of vitamin D and its steroid hormones dictate that these be highly conformationally flexible molecules present a wide variety of shapes to their biologic environments. (b) It is now believed that 1alpha,25(OH)(2)D(3) produces biologic responses through two distinct receptors which recognize totally different shapes of the conformationally flexible 1alpha,25(OH)(2)D(3). Thus, the classic actions of 1alpha,25(OH)(2)D(3) to regulate gene transcription occur as a consequence of the stereospecific interaction of a modified 6-s-trans bowl-shape of 1alpha,25(OH)(2)D(3) with its nuclear receptor (VDR(nuc)). The ability of 1alpha,25(OH)(2)D(3) to generate a variety of rapid (seconds to minutes) biologic responses (opening of chloride channels, activation of PKC and MAP kinases) requires a planar 6-s-cis ligand shape which is recognized by a putative plasma membrane receptor (VDR(mem)) to initiate appropriate signal transduction pathways. This report summarizes the evidence for the specificity of different ligand shapes and the operation of the two receptor families for 1alpha,25(OH)(2)D(3).

Presence of renal 25-hydroxyvitamin-D-1-hydroxylase in species of all vertebrate classes

Abstract 1. 1. Renal 25-hydroxycholecalciferol-1-hydroxylase activity was detected in twenty-five of twenty-eight vertebrate species studied. 2. 2. In general activities these were 2–20 pmole/min per g kidney for normal adult animals and 90–520 pmoles/min per g kidney for animals whose vitamin D intake was restricted prior to measurement of enzyme activity. 3. 3. This is the first demonstration of metabolism of vitamin D by fish tissues.

The 25(OH)D3/1α,25(OH)2D3-24R-hydroxylase: a catabolic or biosynthetic enzyme?

The kidney is the major source of the circulating dihydroxylated metabolites of vitamin D, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. The enzymes which catalyze the production of these two dihydroxylated vitamin D metabolites are the 25(OH)D3-1α-hydroxylase (1α-hydroxylase) and –24R-hydroxylase (24R-hydroxylase), respectively. While there is no controversy regarding the fundamental importance of the 1α-hydroxylase in the production of the steroid hormone 1α,25(OH)2D3, the biologic significance of the 24R-hydroxylase has been the subject of ongoing discussion. Some hold that it is strictly catabolic, leading to side chain oxidation and cleavage of 25-hydroxylated vitamin D sterols, and others hold that it plays a biosynthetic role in the production of 24R,25(OH)2D3 which has biologic activities distinct from those of 1α,25(OH)2D3. The 24R-hydroxylase has properties in common with other multicatalytic steroidogenic enzymes: (1) the enzyme carries out multiple oxidative and carbon-carbon bond cleavages; (2) it utilizes two natural substrates; (3) its regulation varies depending on the cell or tissue in which it occurs. The purpose of this paper is to review the current literature relevant to the characteristics of the 24R-hydroxylase and its regulation in the context of other multicatalytic steroid hydroxylases in order to provide a perspective regarding its possible function as both a catabolic and activating enzyme in the vitamin D endocrine system.

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

Abstract The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D 3 to both 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and 24R,25-dihydroxyvitamin D 3 [24R,25-(OH) 2 D 3 ] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D 3 metabolites is required for the generation of all the biological responses mediated by the parent vitamin D 3 . In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH) 2 -D 3 on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH) 2 D 3 . We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH) 2 D 3 or 1,25(OH) 2 D 3 singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca 2+ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH) 2 D 3 and 1,25(OH) 2 D 3 are simultaneously required for normalization of calcium homeostasis.

A molecular description of ligand binding to the two overlapping binding pockets of the nuclear vitamin D receptor (VDR): structure-function implications

Molecular modeling results indicate that the VDR contains two overlapping ligand binding pockets (LBP). Differential ligand stability and fractional occupancy of the two LBP has been physiochemically linked to the regulation of VDR-dependent genomic and non-genomic cellular responses. The purpose of this report is to develop an unbiased molecular modeling protocol that serves as a good starting point in simulating the dynamic interaction between 1alpha,25(OH)2-vitamin D3 (1,25D3) and the VDR LBP. To accomplish this goal, the flexible docking protocol developed allowed for flexibility in the VDR ligand and the VDR atoms that form the surfaces of the VDR LBP. This approach blindly replicated the 1,25D3 conformation and side-chain dynamics observed in the VDR X-ray structure. The results are also consistent with the previously published tenants of the vitamin D sterol (VDS)-VDR conformational ensemble model. Furthermore, we used flexible docking in combination with whole-cell patch-clamp electrophysiology and steroid competition assays to demonstrate that (a) new non-vitamin D VDR ligands show a different pocket selectivity when compared to 1,25D3 that is qualitatively consistent with their ability to stimulate chloride channels and (b) a new route of ligand binding provides a novel hypothesis describing the structural nuances that underlie hypercalceamia.