Mineral regulation of vitamin D metabolism

The pediatrician's interest in vitamin D metabolism stems from the once-endemic rachitic deformities induced by vitamin D deficiency; later, clinical research of inherited forms of rickets established further principles of vitamin D metabolism and action. Constantine Anast, as both clinician and researcher, maintained an enthusiastic interest in vitamin D metabolism. His investigative esprit fostered my interest, as a fellow in his laboratory, in the synthetic pathway of active vitamin D.The best known active metabolite of vitamin D, 1,25(OH)₂D, is formed by 1βhydroxylation of 25(OH)D, the most abundant circulating form of the vitamin. This well-characterized biochemical conversion is the rate-limiting reaction in the synthesis of 1,25(OH)₂D [1]. The classical homeostatic role of 1,25(OH)₂D is predominantly that of a calcemie agent, an action largely resulting from the metabolite's stimulation of intestinal transport of calcium [2]. Intestinal phosphorus transport, to a lesser extent than calcium transport can be stimulated by 1,25(OH)₂D [3]. Furthermore, skeletal [4] and perhaps renal activity [5] of 1,25(OH)₂D can increase circulating concentrations of calcium. These in vivo effects of 1,25(OH)₂D on mineral homeostasis raise the question of whether feedback control, via mineral regulation of 1,25(OH)₂D production, exists, and the significant mechanisms involved. Here, I will briefly review evidence from earlier studies supporting the notion of calcium and phosphorus regulation of 1α-hydroxylase activity, and present data generated in collaboration with Dr Anast examining vitamin D metabolism in magnesium deficiency.     

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

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 D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. 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)₂D₃ or 1,25(OH)₂D₃ 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 Ca²⁺ 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)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

Vitamin D seco-steroids: unique molecules with both hormone and possible membranophilic properties.

The mode of action of vitamin D₃ (a seco steroid with a broken B ring) for calcium and phosphorus homeostasis is mediated at least in part through its metabolic transformation to 1, 25-dihydroxyvitamin D and 24, 25-dihydroxyvitamin D₃. A vitamin D endocrine system produces in a carefully regulated fashion amounts of 1, 25(OH)₂D₃dictated by the calcium needs of the organism. 1, 25(OH)₂D₃ is known to induce the biosynthesis of a a calcium binding protein in the intestine and kidney via a mechanism similar to that of classic steroid hormones. Evidence for biological effects specifically generated by 24, 25(OH)₂D₃ D have also been obtained. Definite evidence for the further metabolism of these dihydroxylated metabolites exists; it probably occurs via side chain cleavage with the generation of several new molecules of unknown function. Evaluation of the unique structural aspects of these putative vitamin D metabolites suggests a second set of chemical and biological properties which could link these compounds structurally to the membrane-active polyene antibiotic filipin. These properties may rationalize some reported “rapid biological effects” pertaining to vitamin D and metabolites which hitherto have been difficult to explain in terms of classical steroid hormone theory. A key unresolved question relates to identification of the evolutionary pathways which resulted in the selection of vitamin D seco-steroids rather than classical (with intact rings) steroids for Ca and P homeostasis.     

1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)₂D₃ and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)₂D₃ altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)₂D₃-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)₂D₃. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)₂D₃ in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Opposing actions of methylxanthines and dibutyryl cyclic AMP on 1,25 dihydroxyvitamin D3 production and calcium fluxes in isolated chick renal tubules

In contrast to dibuturyl cyclic AMP, the methylxanthine phosphodiesterase inhibitors theophylline and caffeine were found to inhibit the conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃ in isolated renal tubules from vitamin D deficient chicks. This inhibition occurred at concentrations of methylxanthines which were shown to increase renal tubule cyclic AMP levels. No effect of theophylline or caffeine on 25 hydroxyvitamin D₃ metabolism in isolated chick renal mitochondria was detected. Because of a demonstrated inhibitory action of calcium (10 and 20 μmol/l) on renal mitochondrial conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃, the effect of theophylline and dibutyryl cyclic AMP on cellular calcium-45 efflux and total renal tubule calcium content was estimated. Theophylline 10 mmol/l was found to inhibit renal tubular calcium efflux and to increase total cellular calcium content, while dibutyryl cyclic AMP 1 mmol/l had the reverse effect on both parameters. Divergent actions of the methylxanthines and dibutyryl cyclic AMP on the formation of 1,25 dihydroxyvitamin D₃ and renal tubule calcium efflux and content support the hypothesis that intracellular calcium is an important regulator of renal vitamin D metabolism. The results indicate that observed actions of methylxanthines cannot always be ascribed to cyclic AMP accumulation.     

The calcitonin/calcitonin gene related peptide-α gene is not required for 1α,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), can suppress disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Calcium appears to be a critical component of 1,25(OH)₂D₃-mediated suppression of EAE, as complete disease prevention only occurs with a concomitant increase in serum calcium levels. Calcitonin (CT) is a peptide hormone released in response to acute increases in serum calcium, which led us to explore its importance in 1,25(OH)₂D₃-mediated suppression of EAE. Previously, we discovered that co-administration of pharmacological doses of CT enhanced the suppressive effect of 1,25(OH)₂D₃ on EAE, suggesting CT may play a role in 1,25(OH)₂D₃-mediated suppression of EAE. To determine the importance of CT in EAE we have utilized a mouse strain in which the gene encoding CT and its alternative splice product, calcitonin gene related peptide-α (CGRP), have been deleted. Deletion of the CT/CGRP gene had no effect on EAE progression. Furthermore, treatment with 1,25(OH)₂D₃ suppressed EAE in CT/CGRP knock-out mice equal to that in wild type mice. Therefore, we conclude that CT is not necessary for 1,25(OH)₂D₃-mediated suppression of EAE.     

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.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Vitamin D Metabolites and Their Association with Calcium,Phosphorus, and PTH Concentrations,Severity of Illness,and Mortality in Hospitalized Equine Neonates

Background

Hypocalcemia is a frequent abnormality that has been associated with disease severity and outcome in hospitalized foals. However, the pathogenesis of equine neonatal hypocalcemia is poorly understood. Hypovitaminosis D in critically ill people has been linked to hypocalcemia and mortality; however, information on vitamin D metabolites and their association with clinical findings and outcome in critically ill foals is lacking. The goal of this study was to determine the prevalence of vitamin D deficiency (hypovitaminosis D) and its association with serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations, disease severity, and mortality in hospitalized newborn foals.

Methods and Results

One hundred newborn foals ≤72 hours old divided into hospitalized (n = 83; 59 septic, 24 sick non-septic [SNS]) and healthy (n = 17) groups were included. Blood samples were collected on admission to measure serum 25-hydroxyvitamin D₃ [25(OH)D₃], 1,25-dihydroxyvitamin D₃ [1,25(OH) ₂D₃], and PTH concentrations. Data were analyzed by nonparametric methods and univariate logistic regression. The prevalence of hypovitaminosis D [defined as 25(OH)D₃ <9.51 ng/mL] was 63% for hospitalized, 64% for septic, and 63% for SNS foals. Serum 25(OH)D₃ and 1,25(OH) ₂D₃ concentrations were significantly lower in septic and SNS compared to healthy foals (P<0.0001; P = 0.037). Septic foals had significantly lower calcium and higher phosphorus and PTH concentrations than healthy and SNS foals (P<0.05). In hospitalized and septic foals, low 1,25(OH)₂D₃ concentrations were associated with increased PTH but not with calcium or phosphorus concentrations. Septic foals with 25(OH)D₃ <9.51 ng/mL and 1,25(OH) ₂D₃ <7.09 pmol/L were more likely to die (OR=3.62; 95% CI = 1.1-12.40; OR = 5.41; 95% CI = 1.19-24.52, respectively).

Conclusions

Low 25(OH)D₃ and 1,25(OH)₂D₃ concentrations are associated with disease severity and mortality in hospitalized foals. Vitamin D deficiency may contribute to a pro-inflammatory state in equine perinatal diseases. Hypocalcemia and hyperphosphatemia together with decreased 1,25(OH)₂D₃ but increased PTH concentrations in septic foals indicates that PTH resistance may be associated with the development of these abnormalities.     

Influence of ultraviolet B radiation on vitamin D3 metabolism in vitamin D3-resistant New World primates

We investigated the occurrence of rickets in adolescent tamarins (Saguinus imperator) residing at the Los Angeles Zoo. Compared to tamarins in the same colony without clinical evidence of bone disease (N = 6), rachitic platyrrhines (N = 3) had a decrease in their serum calcium concentration (P < .05). The affected tamarins also had lower serum 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) levels than did nonaffected colony mates, but 2–10-fold higher concentrations than in Old World primates of a comparable developmental stage. New World primates in many different genera are known to exhibit target organ resistance to the active vitamin D₃ metabolite, 1,25-(OH)₂D₃, compensated by maintenance of high circulating concentrations of 1,25-(OH)₂D₃. The relatively low serum 1,25-(OH)₂D₃ concentration in rachitic tamarins and ultraviolet B radiation deficient environment of these primates suggested that bone disease may be linked to a deficiency in substrate for 1,25-(OH)₂D₃, 25 hydroxyvtamin D₃ (25-OHD₃). Chronic exposure of platyrrhines in three different vitamin D resistant genera to an artificial UVB source resulted in 1) a significant increase in the mean serum 25-OHD₃ (P < .001) and 1,25-(OH)₂D₃ (P < .02) level over that encountered in platyrrhines not exposed to UVB; and 2) prevention of rachitic bone disease in irradiated individuals. These data further show that the serum 25-OHD₃ and 1,25-OH₂D₃ levels are positively correlated in vitamin D-resistant platyrrhines (r = 0.64; P= .0014) and suggest that a compromise in cutaneous vitamin D₃ production by means of UVB deprivation may limit necessary 1,25-(OH)₂D₃ production. © 1992 Wiley-Liss, Inc.     

Vitamin D Up-regulates Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization Mediated by Cystathionine-γ-lyase (CSE) Activation and H2S Formation in 3T3L1 Adipocytes

A scientific explanation for the beneficial role of vitamin D supplementation in the lowering of glycemia in diabetes remains to be determined. This study examined the biochemical mechanism by which vitamin D supplementation regulates glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mm) in the presence or absence of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (25, 50 nm), the active form of vitamin D. 1,25(OH)₂D₃ treatment caused significant up-regulation of GLUT4 total protein expression and its translocation to cell surface, and an increase in glucose uptake as well as glucose utilization in HG-treated cells. 1,25(OH)₂D₃ also caused cystathionine-γ-lyase (CSE) activation and H₂S formation in HG-treated adipocytes. The effect of 1,25(OH)₂D₃ on GLUT4 translocation, glucose utilization, and H₂S formation was prevented by propargylglycine, an inhibitor of CSE that catalyzes H₂S formation. Studies using antisense CSE also demonstrated the inhibition of GLUT4 translocation as well as glucose uptake and utilization in 1,25(OH)₂D₃-supplemented CSE-siRNA-transfected adipocytes compared with controls. 1,25(OH)₂D₃ treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared with either insulin or 1,25(OH)₂D₃ alone in HG-treated adipocytes. 1,25(OH)₂D₃ supplementation also inhibited monocyte chemoattractant protein-1 and stimulated adiponectin secretion in HG-treated adipocytes, and this positive effect was prevented in propargylglycine-treated or CSE-knockdown adipocytes. This is the first report to demonstrate that 1,25(OH)₂D₃ up-regulates GLUT4 translocation and glucose utilization and decreases inflammatory markers, which is mediated by CSE activation and H₂S formation in adipocytes. This study provides evidence for a novel molecular mechanism by which 1,25(OH)₂D₃ can up-regulate the GLUT4 translocation essential for maintenance of glucose metabolism.     

Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Studies on the mode of action of calciferol. X. 24-Nor-25-hydroxyvitamin D3, an analog of 25-hydroxyvitamin D3 having "anti-vitamin" activity.

24-Nor-25-hydroxyvitamin D₃, an analog of 25-hydroxyvitamin D₃, has been chemically synthesized in six steps. This steroid was tested in chicks, $\text{in vivo}$, for its ability to generate the classic vitamin D mediated responses of stimulation of intestinal calcium transport and bone calcium mobilization. Although the 24-nor-25-OH-vitamin D₃ itself exhibited no biological activity in these assays, the analog was found to inhibit the normal responses produced by a physiological dose of vitamin D₃. These results suggest that 24-nor-25-OH-vitamin D₃ may satisfy certain requirements expected of a calciferol “anti-vitamin.”