1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture.

The major circulating metabolite of vitamin D3, 25-hydroxycholecalciferol [25(OH)D], has a remarkably long half-life in blood for a (seco)steroid. Data from our studies and others are consistent with the hypothesis that there is a role for skeletal muscle in the maintenance of vitamin D status. Muscle cells internalise vitamin D-binding protein (DBP) from the circulation by means of a megalin/cubilin plasma membrane transport mechanism. The internalised DBP molecules then bind to actin and thus provide an intracellular array of high affinity binding sites for its specific ligand, 25(OH)D. There is evidence that the residence time for DBP in muscle cells is short and that it undergoes proteolytic degradation, releasing bound 25(OH)D. The processes of internalisation of DBP and its intracellular residence time, bound to actin, appear to be regulated. To explore whether 1,25-dihydroxycholecalciferol (calcitriol) has any effect on this process, cell cultures of myotubes and primary skeletal muscle fibers were incubated in a medium containing 10-10M calcitriol but with no added DBP. After 3h pre-incubation with calcitriol, the net uptake of 25(OH)D by these calcitriol-treated cells over a further 4h was significantly greater than that in vehicle-treated control cells. This was accompanied by a significant increase in intracellular DBP protein. However, after 16h of pre-incubation with calcitriol, the muscle cells showed a significantly depressed ability to accumulate 25(OH)D compared to control cells over a further 4 or 16hours. These effects of pre-incubation with calcitriol were abolished in fibers from VDR-knockout mice. The effect was also abolished by the addition of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride channel opening. Incubation of C2 myotubes with calcitriol also significantly reduced retention of previously accumulated 25(OH)D after 4 or 8h. It is concluded from these in vitro studies that calcitriol can modify the DBP-dependent uptake and release of 25(OH)D by skeletal muscle cells in a manner that suggests some inducible change in the function of these cells.

S-25-hydroxyvitamin D and C3-epimers in pregnancy and infancy: An Odense Child Cohort study.

BACKGROUND: Analysis of serum 25-hydroxyvitamin D (s-25(OH)D) may be complicated by the less active or in-active vitamin D metabolite C3-epi-25(OH)D3 (C3-epimer). We aimed to explore the relationship between s-C3-epimer and s-25(OH)D and other determinants and describe the longitudinal course of the C3-epimer fraction in paired mother-child samples. METHOD: S-25(OH)D and s-C3-epimer were estimated by liquid chromatography mass spectrometry in 290 mother-infant pairs from the population-based Odense Child Cohort. Longitudinal analyses were feasible in two subcohorts; B) early and late pregnancy, cord, three and 18months (n=132); and C) early and late pregnancy, delivery and cord (n=105). RESULTS: Mean s-25(OH)D was 50.6-110.4nmol/L at the six time points. The mean C3-epimer fraction was 10.1% at three months, 1.1%-3.0% at the other time points. In multivariate analyses, the s-C3-epimer correlated with s-25(OH)D (all time points, p<0.001), and season, maternal and infant age and maternal vitamin D supplementation at some time points. The C3-epimer fraction fluctuated between adjacent time points. By cosinor analyses, a season-dependent sinusoidal pattern for s-25(OH)D and C3-epimer fraction was found and changes between adjacent time points depended on season (p<0.007 or trend). In early infancy, subtraction of the C3-epi-25(OH)D3 from total s-25(OH)D resulted in reclassification of 8% of the children by use of the 75nmol/L cut off for s-25(OH)D. CONLCUSION: The s-C3-epimer was independently correlated to s-25(OH)D, season, maternal vitamin D supplementation, maternal and infant age. The C3-epimer fraction was only of clinical importance in early infancy, where it could lead to misclassification of the vitamin D status.

Vitamin D in cutaneous carcinogenesis: part I.

Skin cancer is the most common cancer in the United States. Exposure to ultraviolet radiation is a known risk factor for skin cancer but is also the principal means by which the body obtains vitamin D. Several studies have suggested that vitamin D plays a protective role in a variety of internal malignancies. With regard to skin cancer, epidemiologic and laboratory studies suggest that vitamin D and its metabolites may have a similar protective effect. These noncalcemic actions of vitamin D have called into question whether the current recommended intake of vitamin D is too low for optimal health and cancer prevention. Part I will review the role of vitamin D in the epidermis; part II will review the role of vitamin D in keratinocyte-derived tumors to help frame the discussion on the possible role of vitamin D in the prevention of skin cancer.

A comparison of vitamin D activity in paired non-malignant and malignant human breast tissues.

Links between a low vitamin D status and an increased risk of breast cancer have been observed in epidemiological studies. These links have been investigated in human tissue homogenates and cultured cell lines. We have used non-malignant, malignant and normal reduction mammoplasty breast tissues to investigate the biological and metabolic consequences of the application of vitamin D to intact ex vivo human breast tissue. Tissues were exposed to 1α,25(OH)(2)D(3) (1,25D; active metabolite) and 25(OH)D (25D; pre-metabolite). Changes in mRNA expression and protein expression after vitamin D exposure were analysed. Results indicate that while responses in normal and non-malignant breast tissues are similar between individuals, different tumour tissues are highly variable with regards to their gene expression and biological response. Collectively, malignant breast tissue responds well to active 1,25D, but not to the inactive pre-metabolite 25D. This may have consequences for the recommendation of vitamin D supplementation in breast cancer patients.

The association of serum 25-hydroxyvitamin D3 and D2 with depressive symptoms in childhood--a prospective cohort study.

BACKGROUND: Depression in adolescence is common and early onset predicts worse outcome in adulthood. Studies in adults have suggested a link between higher total 25-hydroxyvitamin D [25(OH)D] concentrations and lower risk of depression. OBJECTIVES: To investigate (a) the association between serum 25(OH)D(2) and 25(OH)D(3) concentrations and depressive symptoms in children, and (b) whether the associations of 25(OH)D(2) and 25(OH)D(3) are different from, and independent of, each other. METHODS: Prospective cohort study with serum 25(OH)D(2) and 25(OH)D(3) concentrations measured at mean age of 9.8 years and depressive symptoms assessed with the Mood and Feelings Questionnaire by a trained interviewer at the mean ages of 10.6 years (n = 2,759) and 13.8 years (n = 2,752). RESULTS: Higher concentrations of 25(OH)D(3) assessed at mean age 9.8 years were associated with lower levels of depressive symptoms at age 13.8 years [adjusted risk ratio (RR; 95% confidence interval (CI)): 0.90 (0.86-0.95)], but not at age 10.6 years [adjusted RR (95% CI): 0.98 (0.93-1.03)] and with increased odds of decreasing symptoms between age 10.6 and 13.8 years [adjusted RR (95% CI): 1.08 (1.01-1.16)]. Serum 25(OH)D(2) concentrations were not associated with depressive symptoms. CONCLUSIONS: This is the first study in children to suggest that the association between 25(OH)D(3) concentrations and depression emerges in childhood. The association is independent of a wide range of potential confounding factors, and appears to be stronger with greater time separation between assessment of 25(OH)D(3) and assessment of depressive symptoms. Confirmation of our findings in large prospective studies and trials would be valuable.

Evolution of the calcium paradigm: the relation between vitamin D, serum calcium and calcium absorption.

Osteoporosis is the index disease for calcium deficiency, just as rickets/osteomalacia is the index disease for vitamin D deficiency, but there is considerable overlap between them. The common explanation for this overlap is that hypovitaminosis D causes malabsorption of calcium which then causes secondary hyperparathyroidism and is effectively the same thing as calcium deficiency. This paradigm is incorrect. Hypovitaminosis D causes secondary hyperparathyroidism at serum calcidiol levels lower than 60 nmol/L long before it causes malabsorption of calcium because serum calcitriol (which controls calcium absorption) is maintained until serum calcidiol falls below 20 nmol/L. This secondary hyperparathyroidism, probably due to loss of a "calcaemic" action of vitamin D on bone first described in 1957, destroys bone and explains why vitamin D insufficiency is a risk factor for osteoporosis. Vitamin D thus plays a central role in the maintenance of the serum (ionised) calcium, which is more important to the organism than the preservation of the skeleton. Bone is sacrificed when absorbed dietary calcium does not match excretion through the skin, kidneys and bowel which is why calcium deficiency causes osteoporosis in experimental animals and, by implication, in humans.

The effect of 25-hydroxyvitamin D3 on the immune system.

AIM: To examine the effect of 25-OH vitamin D3 on the immune system in patients with nutritional rickets. METHODS: Fifty-three patients were included in our study between April 2002 and March 2004. Diagnosis of rickets was based on clinical, biochemical and radiological examinations. Cell surface markers (CD), complement factors (C), and immunoglobulin (Ig) levels were determined to find out any relationship between rickets and immune system deficiency. RESULTS: Among the causes of admission to hospital, fever (66%) and coughing (62.2%) were the most frequent. Pneumonia was accompanied by rickets in 47.1% of the cases. Plasma CD4 levels before the treatment were higher than those in the post-treatment period, whereas CD20 and CD56 levels were lower. CONCLUSION: B cell and natural killer cell reduction which occur because of vitamin D deficiency may contribute to the development of pneumonia and other infections in patients with nutritional rickets.

Vitamin D, aging, and cancer.

Deficiency of the prohormone calcidiol (25OH vitamin D(3)) seems to be associated with several aging-related chronic diseases including cancer. Our results suggest that calcidiol is mainly responsible for differentiation homeostasis, whereas calcitriol might be more involved in calcium homeostasis. Therefore, an imbalance of calcidiol rather than calcitriol is a risk factor for cancer and chronic diseases. Calcidiol insufficiency, as well as insufficient solar exposure, is associated with increased risk of several solid cancers. Both a vitamin D(3) deficiency and a high concentration of calcidiol may increase cancer risk. Similarly, aging phenomena show a U-shaped association with vitamin D bioactivity. Therefore, the chronic diseases and cancers related to aging might be prevented by an optimal concentration of serum calcidiol, which remains to be determined.

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells.

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D(3) [25(OH)D(3)]-induced (45)Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65 pM bPTH(1-34) promoted enhanced (45)Ca uptake between 1 and 10 min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50-550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D(3) resulted in a slower stimulation of (45)Ca uptake 5-10 min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10 min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence--that after steroid --streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.

Relationship between 25-(OH) D3, the IGF-I system, leptin, anthropometric and body composition variables in a healthy, randomly selected population.

OBJECTIVE: This study prompted us to investigate the relationship between 25-(OH) D (3) and the IGF-I system, leptin, sex, age, anthropometric and body composition variables in healthy adults. We hypothesised that these variables would regulate 25-(OH) D (3) concentrations. DESIGN: We included 253 subjects--126 men and 127 women. Body mass index (BMI) and body composition was determined, along with serum leptin, total IGF-I, free IGF-I, IGFBP3 and plasma 25-(OH) D (3) concentrations. RESULTS: 25-(OH) D (3) deficiency was observed in 69 subjects. There was a difference between 25-(OH) D (3) values and season (summer vs. winter). We observed similar 25-(OH) D (3) concentrations in men to those in women. The differential characteristics in subjects without 25-(OH) D (3) deficiency were lower BMI, fat mass and body fat and higher free IGF-I. We observed that leptin increased in the last decades and IGF-I system decreased by decade in both men and women. In subjects without 25-(OH) D (3) deficiency, there was a correlation between free IGF-I and 25-(OH) D (3) in men, and a negative correlation between 25-(OH) D (3) and age, BMI, fat mass and leptin and a positive correlation with total IGF-I in women. The multivariate linear regression analysis explained 37.8 % of 25-(OH) D (3) variability in men and 39 % in women, and only season and free IGF-I made an independent contribution to 25-(OH) D (3) in men, and season and fat mass in women. CONCLUSION: These data suggest that free IGF-I in men and fat mass in women could regulate 25-(OH) D (3) concentrations.

The role of Vitamin D3 metabolism in prostate cancer.

Vitamin D deficiency increases risk of prostate cancer. According to our recent results, the key Vitamin D hormone involved in the regulation of cell proliferation in prostate is 25(OH) Vitamin D3. It is mainly acting directly through the Vitamin D receptor (VDR), but partially also through its 1alpha-hydroxylation in the prostate. A deficiency of 25(OH) Vitamin D is common especially during the winter season in the Northern and Southern latitudes due to an insufficient sun exposure, but Vitamin D deficient diet may partially contribute to it. A lack of Vitamin D action may also be due to an altered metabolism or Vitamin D resistance. Vitamin D resistance might be brought up by several mechanisms: Firstly, an increased 24-hydroxylation may increase the inactivation of hormonal Vitamin D metabolites resulting in a Vitamin D resistance. This is obvious in the cancers in which an oncogenic amplification of 24-hydroxykase gene takes place, although an amplification of this gene in prostate cancer has not yet been described. During the aging, the activity of 24-hydroxylase increases, whereas 1alpha-hydroxylation decreases. Furthermore, it is possible that a high serum concentration of 25(OH)D3 could induce 24-hydroxylase expression in prostate. Secondly, Vitamin D receptor gene polymorphism or defects may result in a partial or complete Vitamin D resistance. Thirdly, an overexpression or hyperphosphorylation of retinoblastoma protein may result in an inefficient mitotic control by Vitamin D. Fourthly, endogenous steroids (reviewed by [D.M. Peehl, D. Feldman, Interaction of nuclear receptor ligands with the Vitamin D signaling pathway in prostate cancer, J. Steroid Biochem. Mol. Biol. (2004)]) and phytoestrogens may modulate the expression of Vitamin D metabolizing enzymes. In summary, the local metabolism of hormonal Vitamin D seems to play an important role in the development and progression of prostate cancer.

The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients.

The aim of this study was to evaluate the effect of vitamin D3 supplementation on insulin secretion and insulin resistance. Ten females with type 2 diabetes being treated with oral hypoglycaemic agents and with normal serum and urine calcium levels were enrolled in the study. The study was conducted in March, when levels of vitamin D are lowest in our region. The level of plasma 25(OH)D was measured (normal range in winter 25-120 nmol/l). The first (FPIS) and second (SPIS) phases of insulin secretion were studied during IVGTT. Peripheral insulin resistance was measured. A group of 17 age- and BMI-matched females with normal glucose tolerance served as a control group. The diabetic patients were treated with cholecalciferol 1332 IU daily for one month. The mean plasma 25(OH)D level was 35.3 +/- 15.1 nmol/l at baseline, 70% of patients being vitamin D deficient. After one month of treatment with vitamin D3, the plasma 25(OH)D level increased by a mean of 75.8%; 70% of the patients achieved normal vitamin D levels. FPIS increased significantly by 34.3%, while the change in SPIS of 20.4% was not significant (p > 0.8). We found a significant correlation between the change in FPIS and the change in 25(OH)D level after vitamin D3 supplementation (p < 0.018). The results showed a decrease of 21.4% in insulin resistance after one month, but the change was not significant. Bearing in mind that the main defects in type 2 diabetes mellitus are reduced FPIS and insulin resistance, and the favourable effect vitamin D3 had on them, we suggest vitamin D3 deficiency may at least partly contribute to the impairment of insulin secretion and probably of insulin action. Our results suggest that vitamin D3 supplementation could be an element in the complex treatment of type 2 diabetes mellitus during the winter.

Vitamin D control of osteoblast function and bone extracellular matrix mineralization.

Vitamin D is the major regulator of calcium homeostasis and protects the organism from calcium deficiency via effects on the intestine, kidney, parathyroid gland, and bone. Disturbances in the vitamin D endocrine system (e.g., vitamin D-dependent rickets type I and type II), result in profound effects on the mineralization of bone. Recent studies with vitamin D receptor knockout mice also show effects on bone. It is questioned whether vitamin D has a direct effect on bone formation and mineralization. In rickets and particular vitamin D receptor knockout mice, calcium supplementation restores bone mineralization. However, the vitamin D receptor is present in osteoblasts, and vitamin D affects the expression of various genes in osteoblasts. This review focuses on the role of vitamin D in the control of osteoblast function and discusses the current knowledge of the direct effects of vitamin D on mineralization. Moreover, the role of vitamin D metabolism and the mechanism of action of vitamin D and interaction with other hormones and factors are discussed.

Role of calciotrophic hormones in calcium mobilization of lactation.

The postpartum period can be a time when profound changes in calcium metabolism and bone mineral density (BMD) occur, particularly in association with lactation. We investigated the hypothesis that calciotrophic hormones [1,25-dihydroxyvitamin D, 25-hydroxyvitamin D, and parathyroid hormone (PTH)] are different by lactation practice or hormone status [PTH-related peptide (PTHrP), estradiol, and prolactin] and have a potential role in the bone loss and recovery associated with lactation. 1,25-Dihydroxyvitamin D, 25-hydroxyvitamin D, PTH, femoral BMD, PTHrP, prolactin, estradiol, and bone turnover markers were measured at 2 wk and at 2, 4, 6, 12, and 18 mo postparturition in 115 postpartum women aged 20-40 y (parity: 0-1). Lumbar spine BMD was measured at 2 wk and at 6, 12, and 18 mo during the postpartum period. PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D concentrations were nonlinear across the 18-mo postpartum period. Between baseline and 18 mo postparturition, PTH and 1,25-dihydroxyvitamin D concentrations did not decline, while there was a substantial decline in 25-hydroxyvitamin D concentrations. PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D concentrations did not differ by lactation practice or by PTHrP, estradiol, or prolactin status. These classic calciotrophic hormones were not associated with concentrations of bone turnover markers or changes in BMD in lactating women. In summary, patterns of change in calciotrophic hormones (PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D) in the 18-mo postpartum period appeared to be independent of PTHrP, estradiol, prolactin, or lactation status and were not associated with bone turnover markers. These data do not support the hypothesis that these three calciotrophic hormones are a central part of the calcium mobilization associated with the bone loss of lactation.

25-hydroxycholecalciferol in poultry nutrition.

Vitamin D is a complex of secosteroids that must undergo metabolic alterations to reach optimal biological activity. The parent compounds 1) ergocalciferol (D2) and 2) cholecalciferol (D3) can be synthesized in the leaves of many plants or in the skin of most animals, respectively. Transport of vitamin D steroids after absorption is associated with vitamin D binding proteins (DBP). In general, the relative binding affinities of the vitamin D steroids are: 25-hydroxy vitamin D3 [25-(OH)D3] = 24,25-dihydroxy vitamin D3 [24,25-(OH)2D3] = 25,26-dihydroxy vitamin D3 [25,26-(OH)2D3] > 25-hydroxy vitamin D2 (25-(OH)D2) > 1,25-dihydroxy vitamin D3 [1,25-(OH)2D3] > vitamin D3. The DBP in poultry does not bind D2 forms effectively, and therefore poultry can not use this form of vitamin D adequately. The concentration of 25-(OH)D3 in blood seems to be well correlated with dietary vitamin D intake or exposure to ultraviolet light. The 1 alpha hydroxylase enzyme in the kidney is subject to negative feedback regulation and is critical for formation of the active metabolite 1,25-(OH)2D3. The intracellular vitamin D receptor (VDR) specifically binds 1,25-(OH)2D3 and is necessary for cellular action. Increased levels of two to three orders of magnitude are required for 25-(OH)D3 to compete with 1,25-(OH)2D3 for binding on VDR. Feeding studies with 25-(OH)D3 suggest it has nearly twice the activity of vitamin D3. Hatchability studies have shown that 25-(OH)D3 supports good fertility and hatchability, whereas hens fed only 1,25-(OH)2D3 did not have normal hatchability. Likewise, 1,25-(OH)2D3 seems to reach toxic levels at dietary concentrations only two to three times optimal dietary levels whereas feeding 25-(OH)D3 for extended periods at levels 8 to 10 times requirement seems to have no adverse effects. It seems that 25-(OH)D3 is the most active metabolite of vitamin D3, ultimately capable of supporting both cellular functions and embryonic development in chickens and turkeys when fed as the sole source of vitamin D3.

Hormones, peripherally activated prohormones and regulation of the Th1/Th2 balance.

There is much interest in the factors that control the cytokine profile of T-helper (Th) lymphocytes, and attention has focused on feedback from the cytokines themselves. In general, Th1 cytokines promote Th1 activity and inhibit Th2 activity, and vice versa. Both Th1 and Th2 responses should therefore be stable. However, in vivo, many responses start predominantly as Th1 and then shift to Th2. Why do they do this? As discussed here, an important influence on this shift that has been largely ignored in in vitro work is the endocrine system.