Role of vitamin D derivatives in intestinal tissue of patients with inflammatory bowel diseases

Background and aimThe adhesion molecule expression and matrix metalloproteinases (MMPs) are proposed to be major factors for intestinal injury mediated by T cells in (IBD) and are up-regulated in intestinal mucosa of IBD patients. To investigate the effect of vitamin D derivatives on adhesion molecules and MMPs in colonic biopsies of IBD patients.MethodsBiopsies from inflamed and non-inflamed tract of terminal ileum and colon and PBMC from the same IBD patients were cultured with or without vitamin D derivatives. MMP activity and adhesion molecule levels were determined.Results1,25(OH)₂D₃ and ZK 191784 significantly decrease ICAM-1 protein levels in the biopsies obtained only from the inflamed region of intestine of UC patients, while MAdCAM-1 levels decrease in the presence of 1,25(OH)₂D₃ in the non-inflamed region, and, in the presence of ZK, in the inflamed one. In CD patients 1,25(OH)₂D₃ and ZK decrease ICAM-1 and MAdCAM-1 in the biopsies obtained from the non-inflamed and inflamed regions, with the exception of ICAM-1 in the inflamed region in the presence of 1,25(OH)₂D₃. The expression of MMP-9, MMP-2, and MMP-3 decreases in the presence of vitamin D derivatives in UC and CD with the exception of 1,25(OH)₂D₃ that does not affect the levels of MMP-9 and MMP-2 in CD. Vitamin D derivatives always affect MMP-9, MMP-2 and ICAM-1 in PBMC of UC and CD patients.ConclusionsBased on the increased expression of ICAM-1, MAdCAM-1 and MMP-2,-9,-3 in IBD, our study suggests that vitamin D derivatives may be effective in the management of these diseases.     

Plasma vitamin D metabolite levels in phosphorus deficient rats during the development of vitamin D deficient rickets

Plasma levels of the vitamin D metabolites were related to changes in bone morphology during the development of rickets in rats deprived of phosphorus and vitamin D. Weanling rats were studied at 1, 3, and 5 wk after onset of diets deficient in phosphorus or in both phosphorus and vitamin D. Bone histology and morphometry were carried out and measurements were made of ⁴⁵Ca and ³²P absorption, serum Ca and P, and plasma 25(OH)D₃, 24,25 (OH)₂D₃ and 1,25 (OH)₂D₃. After 1 wk of vitamin D restriction, the plasma levels of 25(OH)D₃ and 24,25(OH)₂D₃ were non-detectable (<0.5 and <0.8 ng/ml). The plasma 1,25(OH)₂D₃ level was elevated at 1 wk (105.5 pg/ml) and fell to 19 pg/ml by 5 wk. At 1 wk mild rachitic lesions in epiphyseal cartilage were observed despite the elevated 1,25(OH)D₃ level. Serum Ca and P levels and values for ⁴⁵Ca and ³²P absorption decreased and the severity of the rickets increased with the fall in plasma levels of 1,25(OH)₂D₃. In vitamin D replete, phosphate deficient rats the epiphyseal cartilage was normal throughout the 5 wk study peroid. Our results provide further evidence that physiological levels of 1,25 (OH)₂D₃ will not prevent rickets without adequate plasma concentrations of either 25(OH)D₃ or 24,25(OH)₂D₃.     

Dysregulation of vitamin D3 synthesis leads to enhanced cholangiocarcinoma growth

BackgroundCholangiocarcinoma is a deadly biliary tumour with limited treatment strategies. Vitamin (1,25(OH)₂D) has anti-proliferative effects on several cancers. Vitamin D₃ is synthesized by the enzyme, CYP27B1, and signals via the nuclear vitamin D₃ receptor. The enzyme, CYP24A1, degrades vitamin D₃.Aims(i) Measure the expression of CYP27B1, CYP24A1, and vitamin D₃ receptor in human nonmalignant and cholangiocarcinoma lines and biopsy control or tumour samples; and (ii) evaluate the effects of vitamin D₃ on vitamin D₃ synthesis and cholangiocarcinoma growth.MethodsIn vitro studies were performed in malignant and nonmalignant cholangiocytes. Vitamin D₃ receptor, CYP24 and CYP27 expression was measured in cell lines and biopsy samples. Cell lines were stimulated with vehicle or vitamin D₃ from 30 min to 48 h. Cell viability was assessed by MTS assays and BrdU incorporation. Vitamin D₃ receptor, CYP24A1 and CYP27B1 expression was measured in cholangiocarcinoma cells stimulated with vehicle or vitamin D₃.ResultsIn cholangiocarcinoma lines and biopsy samples, vitamin D₃ receptor and CYP24A1 expression increased compared to controls, whereas CYP27B1 expression was decreased or unchanged. Vitamin D₃ induced nuclear translocation of vitamin D₃ receptor in cholangiocarcinoma and decreased cholangiocarcinoma growth.ConclusionTreatment with vitamin D₃ decreased CYP24A1, whereas CYP27B1 expression increased. Modulation of vitamin D₃ synthesis may be important in the management of cholangiocarcinoma.     

Decreased expression of vitamin D receptor may contribute to the hyperimmune status of patients with acquired aplastic anemia

Acquired aplastic anemia (AA) is an immune‐mediated bone marrow failure syndrome. 1α,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). Previous studies have established that 1,25(OH)₂D₃ and VDR were involved in the pathogenesis of some autoimmune diseases. In this study, we evaluated the involvement of 1,25(OH)₂D₃ and VDR on T‐cell responses in AA. Plasma 25(OH)D₃ levels were comparable between patients with AA and healthy controls. Surprisingly, VDR mRNA was significantly lower in untreated patients with AA than in healthy controls. Subsequent in vitro experiments revealed that 1,25(OH)₂D₃ treatment suppressed the proliferation of lymphocytes and inhibited the secretion of interferon‐γ, tumor necrosis factor‐α, and interleukin‐17A, meanwhile promoting the production of transforming growth factor‐β1 in patients with AA. Moreover, 1,25(OH)₂D₃ inhibited the differentiation of type 1 and Th17 cells but induced the differentiation of type 2 and regulatory T cells. Interestingly, VDR mRNA was elevated in healthy controls after 1,25(OH)₂D₃ treatment, but not in patients with AA. In conclusion, decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in patients with AA.     

The physiologic significance of plasma transport of vitamin D and metabolites

Vitamin D and its metabolites do not circulate free but are bound to specific plasma transport proteins that solubilize them and protect them from oxidative inactivation. A specific vitamin D transport protein has been demonstrated in man, rat and chick; it preferentially binds 25-hydroxyvitamin D (25-(OH)D), the major active form of the vitamin in the circulation. The binding of vitamin D and its analogs by the D/25-(OH)D transport protein from rat plasma involves recognition of structural properties and steric configuration of both the single ring and the side chain. The C-25 hydroxyl group plus the vitamin D₃ configuration of both the side chain and the single ring maximize the binding. Although the binding of vitamin D₂ and its analogs by human and rat D/25-(OH)D transport protein is roughly equivalent to that of vitamin D₃ and its analogs, abnormal plasma binding of vitamin D₂ and 25-(OH)D₂ by the D/25-(OH)D transport protein in chick plasma may play an important role in the increased metabolic inactivation of these compounds and the resultant decreased antirachitic potency of vitamin D₂ in the chick. Preliminary studies suggest that 1,25-(OH)₂D is carried by the D/25-(OH)D transport protein in rat plasma but that a specific dihydroxy transport protein also exists in human plasma. These studies suggest that transport proteins play an important role in the facilitation of vitamin D transport and normal vitamin D metabolism.     

p38 MAP kinase activity is correlated with angiotensin II type 1 receptor blocker-induced left ventricular reverse remodeling in spontaneously hypertensive heart failure rats

BackgroundAngiotensin II type 1 receptor blocker L-158,809 (ARB) induces reverse left ventricular (LV) remodeling in spontaneously hypertensive heart failure (SHHF) rats. However, the signaling mechanism that mediates ARB-induced reverse LV remodeling remains unclear. The present study was to determine if changes in mitogen-activated protein kinase (MAPK, including ERK, JNK, and p38) signaling correlate with ARB-elicited reversal of cardiac hypertrophy in SHHF rats.Methods and ResultsIn 1 set of experiments, 5-month-old lean female SHHF rats were treated with L-158,809 (ARB) or the vasodilator hydralazine (HYD) for 1 month, respectively. In a second set of experiments, 5-month-old SHHF rats were treated with ARB for 6 months or 1 month and then with HYD for 5 months. Either ARB or HYD normalized left ventricular end systolic pressure in SHHF rats relative to normotensive control Wistar Furth (WF) rats at both 6 and 11 months of age, but only ARB reduced heart-to-body weight ratio in SHHF rats to control level. Western blot analysis showed that cardiac p38 MAPK activity was markedly increased in 6-month-old SHHF rats, but dramatically reduced in 11-month-old SHHF rats compared with WF rats, as indicated by the levels of phosphorylated form of p38. The alterations in p38 activity were completely reversed by ARB treatment but not by HYD treatment.ConclusionARB restored normal cardiac p38 activity, which coincided with ARB-induced reverse LV remodeling in SHHF rats, suggesting a strong correlation between p38 signaling and cardiac remodeling.     

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10⁻⁷ M, 1,25(OH)₂D₃ for 24 h resulted in a 20–30% decrease in PKI mRNA. 1,25(OH)₂D₃ treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)₂D₃ directly and tissue-specifically downregulates PKI mRNA in the chick kidney.     

Third F. Raymond Keating, Jr., Memorial Symposium--parathyroid hormone, calcitonin and vitamin D: clinical considerations. II. Vitamin D--1973

The recent advances in our understanding of the functional metabolism of vitamin D to 25-hydroxyvitamin D (25-(OH)D) and subsequently to 1,25-dihydroxyvitamin D (1,25-(OH)₂D) are presented with a review of current views on the regulation of vitamin D metabolism at the 25-hydroxylation and 1-hydroxylation stages. It seems clear that, physiologically, the latter regulation is governed by parathyroid hormone (PTH) under conditions of hypocalcemia and by serum inorganic phosphorus levels under conditions from normal to hypercalcemia. The molecular mechanism of the regulation of the renal hydroxylations remains unknown.The metabolism of 25-(OH)D₃ to 24,25-(OH)₂D₃ and further to 1,24,25-(OH)₃D₃ has been established. The significance of these reactions is unknown, but 1,24,25-(OH)₃D₃ preferentially stimulates intestinal calcium transport.The mechanism whereby 1,25-(OH)₂D₃ stimulates intestinal calcium transport and the receptors of this metabolite in intestine are discussed. In addition, the application of the metabolites of vitamin D to clinical problems has been considered and 1α-(OH)D₃, an important analog of 1,25-(OH)₂D₃, has been introduced as a potentially important therapeutic compound.     

IS 1,25-DIHYDROXY-CHOLECALCIFEROL HARMFUL TO RENAL FUNCTION IN PATIENTS WITH CHRONIC RENAL FAILURE?

Seventeen undialysed adult patients with chronic renal failure took part in a controlled study of the effects of 1,25(OH)₂D₃ and D₃. After a 6-month observation period the patients were allocated at random to two groups for 6 months of treatment with either 1,25(OH)₂D₃ (mean dose 0·5 μg daily) or D₃ (dose 100 μg daily). Treatment was then discontinued and the patients were studied for a further 6 months. Serum iPTH was decreased in both groups but most markedly in the 1,25(OH)₂D₃ group in which the iPTH values became normal. Serum creatinine increased during treatment in both groups. In the group receiving 1,25(OH)₂D₃ this was coupled to an increase in serum calcium within the normal range. Our data demonstrate that 1,25(OH)₂D₃ treatment in patients with chronic renal failure leads to a further reduction in renal function, which may be partially reversible. Physicians should therefore be reluctant to give vitamin D analogues to patients with chronic renal failure unless they have severe symptomatic renal osteodystrophy.     

High prevalence of vitamin D deficiency and its association with left ventricular dilation: An echocardiography study in elderly patients with chronic heart failure

Background and aimsVitamin D deficiency has been associated with chronic heart failure (CHF). We evaluated vitamin D levels in relationship with New York Heart Association (NYHA) classes, N-terminal pro-brain natriuretic peptide (NT-proBNP) values and left ventricular (LV) measures in ≥60 year old patients with stable CHF. Differently from previous investigations, LV function was assessed by transthoracic echocardiography, to provide easily reproducible results.Methods and resultsThe study was performed at geographic latitude 44° N, from March to May and from September to November 2008. Acute HF and diseases or drugs altering vitamin D status were exclusion criteria. NYHA scores and 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D and NT-proBNP concentrations were assessed in 90 (45 F, 45 M) Caucasian patients with CHF secondary to hypertension and/or coronary artery disease. Vitamin D levels were also measured in 31 subjects without heart disease (controls). LV echocardiography was performed in 52 (26 F, 26 M) representative patients. Vitamin D concentrations were significantly lower in CHF cases than in controls. Among subject with CHF, 97.8% presented vitamin D deficiency (25(OH)D < 75 nmol/L), being severe (<25 nmol/L) in 66.7%. LV end-diastolic and end-systolic diameters were significantly longer, LV end-diastolic and end-systolic volumes bigger and fractional shortening lower in CHF patients with 25(OH)D < 25 nmol/L than with 25(OH)D ≥ 25 nmol/L (p < 0.05). Log-values of 25(OH)D were negatively correlated with LV end-systolic diameter and volume (r = ?0.28; p < 0.05). On subgroup analysis, these results persisted only in male patients.ConclusionsIn elderly CHF patients, vitamin D deficiency was highly prevalent and often severe. This first addressed echocardiography study showed a sex-specific association between vitamin D deficiency and LV dilation. Since further echocardiography data are easily obtainable, larger investigations are demanded.     

Vitamin D regulates the tight-junction protein expression in active ulcerative colitis

Objective: Epithelial barrier function is primarily regulated by the tight-junction proteins. Ulcerative colitis (UC) is characterized by Th2 immune response with inflammation and epithelial barrier dysfunction, including an elevation of claudin-2 protein function. Recent studies support an important role of vitamin D in the pathogenesis as well as potential therapy of IBD. Vitamin D deficiency is in fact common in patients with IBD. The aim of the study was to determine whether vitamin D could affect IL-13 and IL-6 levels, and regulate the activity of tight-junction proteins. Claudin-1, -2, -4, and -7 in the inflamed and non-inflamed colonic mucosa of UC patients.

Material and methods: Biopsies from inflamed and non-inflamed tract of colon and rectum from the same active UC patients were cultured with1,25(OH)₂D₃. IL-13, IL-6 and the tight-junction proteins level were determined.

Results: Claudin-1 and claudin-2 proteins were up-regulated in active UC. The treatment with 1,25(OH)₂D₃ decreases the claudin-1 and claudin-2 protein levels in both inflamed and non-inflamed tract. Claudin-4 and claudin-7 proteins were down-regulated and their levels increase after incubation with the 1,25(OH)₂D₃. When the biopsies were incubated with 1,25(OH)₂D₃, a decrease in IL-13 and IL-6 levels was registered.

Conclusions: Our results, indicating the inhibition of cytokine levels and the regulation of claudin-2, claudin-4, and claudin-7 by 1,25(OH)₂D₃, suggest that vitamin D may represent a potential therapeutic agent for the treatment of active UC.     

Cardiac metabolism,inflammation, and peroxisome proliferator-activated receptors modulated by 1,25-dihydroxyvitamin D3 in diabetic rats

Background

High free fatty acid with reduced glucose utilization in diabetes mellitus (DM) impairs cardiac function. Peroxisome proliferator-activated receptors (PPARs) modulate myocardial lipid and glucose homeostasis. The active 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) regulates oxidative stress and inflammation, which may play a key role in the modulation of PPARs. The aim of this study was to investigate whether 1,25(OH)₂D₃ can modulate the cardiac PPARs and fatty acid metabolism.

Methods

Electrocardiogram, echocardiogram, and Western blot analysis were used to evaluate cardiac fatty acid metabolism, inflammation, and PPAR isoform expression in Wistar-Kyoto (WKY) rats, DM rats, and DM rats treated with 1,25(OH)₂D₃.

Results

Compared to healthy rats, DM and 1,25(OH)₂D₃-treated DM rats had lower body weight. DM rats had larger left ventricular end-diastolic diameter, and longer QT interval than healthy or 1,25(OH)₂D₃-treated DM rats. Moreover, compared to healthy or 1,25(OH)₂D₃-treated DM rats, DM rats had fewer cardiac PPAR-α and PPAR-δ protein expressions, but had increased cardiac PPAR-γ protein levels, tumor necrosis factor-α, interleukin-6, 5′ adenosine monophosphate-activated protein kinaseα2, phosphorylated acetyl CoA carboxylase, carnitine palmitoyltransferase 1, PPAR-γ coactivator 1-α, cluster of differentiation 36, and diacylglycerol acyltransferase 2 protein expressions.

Conclusions

1,25(OH)₂D₃ significantly changed the cardiac function and fatty acid regulations in DM hearts, which may be caused by its regulations on cardiac PPARs and proinflammatory cytokines.     

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)₂D₃ and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intronlexon boundarles sequenced. PATIENT A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)₂D₃. MEASUREMENTS Nuclear association of 1,25(OH)₂D₃ was determined in patient and control cells and the functional response to 1,25(OH)₂D₃ was assessed by measurement of 25-hydroxyvitamln D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH), D₃. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5’donor splice site of intron 4. CONCLUSION In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the Characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the + 5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.     

Fok‐I gene polymorphism of vitamin D receptor in patients with beta‐thalassemia major and its effect on vitamin D status

Abstract

Most of the biological actions of vitamin D are mediated by an intracellular receptor (VDR) in which several single nucleotide gene polymorphisms have been identified. Vitamin D deficiency is increasingly identified among thalassemic patients and recent evidence links it with myocardial iron accumulation. The aim of this work was to assess the distribution of the Fok‐I polymorphism of the VDR gene among Greek children and young adults with beta‐thalassemia major and to investigate its association with 25(OH)D₃ and 1,25(OH)₂D₃ serum levels. Sixty‐nine thalassemic patients (35 females and 34 males), with a mean age of 23·05±6·07?years, participated in the study. Genotype frequencies of Fok‐I were similar to those previously reported for other populations; 44·9% of the patients were homozygotes for F allele, 43·5% were heterozygotes and 11·6% were homozygotes for the f allele. Low levels of serum 25(OH)D₃ were recorded, as 41 patients (59·4%) were below the cut‐off limit of 50?nmol/l that determines deficiency, whereas, levels of 1,25(OH)₂D₃ showed wide variability ranging from deficiency (?50?pmol/l) in 34 patients (49·3%) to excess (?125?pmol/l) in 13 patients (18·8%). When stratifying patients according to serum 1,25(OH)₂ D₃ concentrations, a higher prevalence of the f allele was observed in the deficiency group (P?=?0·03). A comparison of the serum concentrations of the two vitamin D metabolites produced a trend towards a negative correlation (r?=??0·204, P?=?0·09). Further studies are required to assess the genetic contribution to the regulation of vitamin D metabolites in the serum of patients with beta‐thalassemia major.     

Vitamin D and human health: lessons from vitamin D receptor null mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.     

Preferential accumulation in vivo of 24R,25-dihydroxyvitamin D(3) in growth plate cartilage of rats

Vitamin D₃ is metabolized in vivo through 25-(OH)D₃ (25D) to both 1α,25-(OH)₂D₃ (1,25D) and 24R,25-(OH)₂D₃ (24,25D). Whereas it is assumed that this metabolism occurs primarily in the kidney, recent studies show that there are extrarenal 1α-and 24R-hydroxylase activities as well, and in chondrocytes, these enzymes are regulated by hormones and growth factors. Furthermore, chondrocytes from the resting zone of growth plate cartilage are a target cell population for 24,25D action, suggesting that this vitamin D metabolite may be targeted to this tissue in vivo. To test this hypothesis, 30 normal male Sprague Dawley rats (120 ±20 g) were divided into three groups of eight animals each, and a control group of six animals, and fed ad libitum for 2 wk, a standard rat chow (Teklad LM-485), which contained 3 IU vitamin D₃/g. The rats were then injected im daily at 9:00am, for 4 consecutive d, with 0.1 mL of either [³H]-25D, [³H]-1,25D or [³H]-24,25D. Each dose contained 13 pmol of hormone (0.36 μCi/dose). The distribution of these metabolites was assessed in tibial bone (B) following ablation of the bone marrow, articular cartilage from the tibia (AC), costochondral growth plate cartilage (GC), serum (S), small intestine (I), and kidney (K). The use of high specific activity tritiated vitamin D metabolites facilitated determining tissue localization and further metabolism without perturbation of the body pools of each major metabolite. Accumulation of [³H]-1,25D or [³H]-24,25D in each tissue was compared to circulating serum levels. In rats dosed with [³H]-25D, the tissue:serum ratios for 1,25D were 4.1 (AC), 35.4 (GC), 1.3 (B), 0.7 (K), and 3.0 (I); and tissue:serum ratios for 24,25D were 1.6 (AC), 9.9 (GC), 0.04 (B), 0.2 (K), and 0.4 (I). In rats dosed with [³H]-24,25D alone, GC was the only tissue to accumulate the administered metabolite at a concentration significantly higher than that of serum. Similarly, in rats dosed with [³H]-1,25D alone, GC was the only tissue to accumulate 1,25D at a concentration higher than that of serum. These results demonstrate, for the first time, that under in vivo conditions, GC specifically accumulates 24,25D and 1,25D. This suggests that growth plate may be a target organ for these two hormones.     

The Effect of Vitamin D on Aldosterone and Health Status in Patients With Heart Failure

BackgroundVitamin D deficiency is associated with heart failure (HF) events, and in animal models vitamin D down-regulates renin-angiotensin-aldosterone system hormones.MethodsPatients with New York Heart Association (NYHA) functional class II–IV HF and a 25OH-D level ≤37.5 ng/mL received 50,000 IU vitamin D₃ weekly (n = 31) or placebo (n = 33) for 6 months. Serum aldosterone, renin, echocardiography, and health status were determined at baseline and 6 months.ResultsMean age of participants was 65.9 ± 10.4 years, 48% were women, 64% were African American, mean ejection fraction was 37.6 ± 13.9%, 36% were in NYHA functional class III, and 64% were in class II. The vitamin D group increased serum 25OH-D (19.1 ± 9.3 to 61.7 ± 20.3 ng/mL) and the placebo group did not (17.8 ± 9.0 to 17.4 ± 9.8 ng/mL). Aldosterone decreased in the vitamin D group (10.0 ± 11.9 to 6.2 ± 11.6 ng/dL) and not in the placebo group (8.9 ± 8.6 to 9.0 ± 12.4 ng/dL; P = .02). There was no difference between groups in renin, echocardiographic measures, or health status from baseline to 6 months. Modeling indicated that variables which predicted change in aldosterone included receiving vitamin D, increasing age, African American race, and lower glomerular filtration rate.ConclusionsVitamin D₃ repletion decreases aldosterone in patients with HF and low serum vitamin D. Vitamin D may be an important adjunct to standard HF therapy. Further study will assess if vitamin D provides long-term benefit for patients with HF.     

Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystal-lographic structure of the glucocorticold and oeltrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations. DESIGN Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25dlhydroxyvltamln D₃ (1,25(OH)₂D₃) and functional responses to this hormone. These cells were also used to Isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patlents was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)₂D₃. Computer analysis of mutations in the VDR gene was carried out using the glucocorticold and oestrogen receptors as model systems. PATIENTS Two patients with HVDRR from unrelated families. MEASUREMENTS Cytosollc binding and nuclear association of 1,25(OH)₂D₃ were determined in control and affected patients, and functional response to 1,25(OH)₂D₃ was assessed by measurement of 2bhydroxyvltamln D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)₂D₃ following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)₂D₃. Sequencing of cDNA revealed single mutations, in patient I (Phe44 → IIe) and in patient II (Lys42 → Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR. CONCLUSIONS Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.