Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH)₂D] in human serum has been developed. Whereas small amounts of [³H]24,25-(OH)₂D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D₃ ([³H]25-OHD₃) can be used as the assay tracer. Since 25-OHD₃ and 24,25-(OH)₂D₃ are equipotent in their competitive displacement of [³H]25-OHD₃ from rat serum, 25-OHD₃ can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH)₂D by batch elution. The purity of biosynthesized [³H]24,25-(OH)₂D₃ and the 24,25-(OH)₂D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH)₂D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH)₂D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.     

Extremely high circulating levels of 1α,25-dihydroxyvitamin D3 in the marmoset,a new world monkey

Compared to most mammals, the marmoset, a new world monkey, requires particularly large amounts of vitamin D to maintain normal growth. We compared serum concentrations of vitamin D metabolites in marmosets with rhesus monkeys and humans. The circulating levels of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] in marmosets were 4 to 10 times higher than those in rhesus monkeys and humans. But none of the marmosets exhibited hypercalcemia. In two marmosets which had suffered bone fractures, the 1α,25-(OH)₂D₃ levels were particularly elevated. These results suggest that the marmoset has an end-organ resistance to 1α,25(OH)₂D₃.     

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.     

Acute Erythemal Ultraviolet Radiation Causes Systemic Immunosuppression in the Absence of Increased 25-Hydroxyvitamin D3 Levels in Male Mice

Vitamin D is synthesised by ultraviolet (UV) irradiation of skin and is hypothesized to be a direct mediator of the immunosuppression that occurs following UV radiation (UVR) exposure. Both UVR and vitamin D drive immune responses towards tolerance by ultimately increasing the suppressive activities of regulatory T cells. To examine a role for UVR-induced vitamin D, vitamin D₃-deficient mice were established by dietary vitamin D₃ restriction. In comparison to vitamin D₃-replete mice, vitamin D₃-deficient mice had significantly reduced serum levels of 25-hydroxyvitamin D₃ (25(OH)D₃, <20 nmol.L⁻¹) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃, <20 pmol.L⁻¹). Following either acute erythemal UVR, or chronic sub-erythemal UVR (8 exposures over 4 weeks) treatment, serum 25(OH)D₃ levels significantly increased in vitamin D₃-deficient female but not male mice. To determine if UVR-induced vitamin D was a mediator of UVR-induced systemic immunosuppression, responses were measured in mice that were able (female) or unable (male) to increase systemic levels of 25(OH)D₃ after UVR. Erythemal UVR (≥4 kJ/m²) suppressed contact hypersensitivity responses (T helper type-1 or -17), aspects of allergic airway disease (T helper type-2) and also the in vivo priming capacity of bone marrow-derived dendritic cells to a similar degree in female and male vitamin D₃-deficient mice. Thus, in male mice, UVR-induced 25(OH)D₃ is not essential for mediating the immunosuppressive effects of erythemal UVR.     

Correlating Pre-Operative Vitamin D Status with Post-Thyroidectomy Hypocalcemia

Objective: To examine the relationship between pre-operative vitamin D status and post-thyroidectomy hypocalcemia.Methods: Retrospective study examining 264 total and completion thyroidectomies conducted between 2007 and 2011. Subjects included had a recorded 25-hydroxyvitamin D (25[OH]D) level within 21 days prior to or 1 day following surgery, did not have a primary parathyroid gland disorder, and were not taking 1,25-dihydroxyvitamin D₃ (calcitriol) prior to surgery. Some subjects were repleted with vitamin D pre-operatively if a low 25(OH)D level (typically below 20 ng/mL) was identified. Pre-operative 25(OH)D, concurrent neck dissection, integrity of parathyroid glands, final pathology, postoperative parathyroid hormone (PTH), calcium nadir and repletion, and length of stay were examined.Results: The mean pre-operative 25(OH)D for all subjects was 25 ng/mL, and the overall rate of post-operative hypocalcemia was 37.5%. Lower pre-operative 25(OH)D did not predict postoperative hypocalcemia (P =.96); however, it did predict the need for postoperative 1,25-dihydroxyvitamin D₃ administration (P =.01). Lower postoperative PTH levels (P =.001) were associated with postoperative hypocalcemia.Conclusion: Pre-operative 25(OH)D did not predict a postoperative decrease in serum calcium, although it did predict the need for 1,25-dihydroxyvitamin D₃ therapy in hypocalcemic subjects. We recommend that 25(OH)D be assessed and, if indicated, repleted pre-operatively in patients undergoing total thyroidectomy.Abbreviations: 25(OH)D = 25-hydroxyvitamin D PTH = parathyroid hormone     

Treatment with 50000 IU Vitamin D2 Every Other Week and Effect on Serum 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and Total 25-Hydroxyvitamin D in Aclinical Setting

ObjectiveTo examine the effect of 50 000 IU-vitamin D₂ supplementation in a clinical setting on serum total 25-hydroxyvitamin D (25[OH]D), 25-hydroxyvitamin D₂ (25[OH]D₂), and 25-hydroxyvitamin D₃ (25[OH]D₃).MethodsThis retrospective cohort study was performed in an urban tertiary referral hospital in Boston, Massachusetts. Patients who had been prescribed 50 000 IU vitamin D₂ repletion and maintenance programs were identified through a search of our electronic medical record. Baseline and follow-up total serum 25(OH)D, 25(OH)D₂, and 25(OH)D₃ levels were compared.ResultsWe examined the medical records of 48 patients who had been prescribed 50 000 IU vitamin D₂ in our clinic. Mean ± standard deviation baseline total 25(OH) D was 31.0 ± 10.6 ng/mL and rose to 48.3 ± 13.4 ng/mL after treatment (P <.001). 25(OH)D₂ increased from 4.2 ± 4.3 ng/mL to 34.6 ± 12.3 ng/mL after treatment (P <.001), for an average of 158 days (range, 35-735 days). Serum 25(OH)D3 decreased from 26.8 ± 10.8 ng/mL to 13.7 ± 7.9 ng/mL (P <.001).ConclusionsFifty thousand IU vitamin D₂ repletion and maintenance therapy substantially increases total 25(OH)D and 25(OH)D2 despite a decrease in serum 25(OH)D3. This treatment program is an appropriate and effective strategy to treat and prevent vitamin D deficiency.(Endocr Pract. 2012;18:399-402)     

1,25-Dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein

1,25-dihydroxyvitamin D₃ increases serum levels of bone Gla protein (BGP). The maximal increase occurs 12 h after injection and is given by 350 ng 1,25(OH)₂D₃ per 180 g body weight. In both 2 and 11 month-old male rats, the maximal increase is about 3 times the normal level, while in 2 month old female rats, the maximal increase is 2 times the normal level. These effects of 1,25(OH)₂D₃ in rats parallel the previously described effects of the vitamin on BGP secretion by rat osteosarcoma cells in culture.BGP is the first bone-specific protein whose synthesis in animals is dramatically increased by 1,25(OH)₂D₃. The possible functions of BGP in the biological actions of 1,25(OH)₂D₃ on bone are discussed.     

Protection against cellular stress by 25‐hydroxyvitamin D3 in breast epithelial cells

25‐Hydroxyvitamin D₃ (25(OH)D₃) is a prohormone and a major vitamin D metabolite. The discovery of (25(OH)D₃) 1α‐hydroxylase in many vitamin D target organs has yielded an increased interest in defining the role(s) of 25(OH)D₃ in these tissues. The etiology of cancer appears to be complex and multi‐factorial. Cellular stress (e.g., DNA damage, hypoxia, oncogene activation) has been identified as one of the key factors responsible for initiating the carcinogenesis process. In this study, we investigated whether 25(OH)D₃ protects breast epithelial cells from cellular stress using an established breast epithelial cell line MCF12F. To better elucidate the role of 25(OH)D₃ in the stress response, we used multiple in vitro stress models including serum starvation, hypoxia, oxidative stress, and apoptosis induction. Under all these stress conditions, 25(OH)D₃ (250 nmol/L) treatment significantly protected cells against cell death. Low‐serum stress induced p53 expression accompanied with downregulation of PCNA, the presence of 25(OH)D₃ consistently inhibited the alteration of p53 and PCNA, suggesting that these molecules were involved in the stress process and may be potential target genes of 25(OH)D₃. miRNA microarray analysis demonstrated that stress induced by serum starvation caused significant alteration in the expression of multiple miRNAs including miR182, but the presence of 25(OH)D₃ effectively reversed this alteration. These data suggest that there is a significant protective role for 25(OH)D₃ against cellular stress in the breast epithelial cells and these effects may be mediated by altered miRNA expression. J. Cell. Biochem. 110: 1324–1333, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

Isolation,identification, and biological activity of 23,25,26-trihydroxyvitamin D3, an in vitro and in vivo metabolite of vitamin D3

Kidney homogenates from vitamin D₃-supplemented chicks incubated with 25-hydroxyvitamin D₃ [25(OH)D₃] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D₃ [23,25,26(OH)₃D₃] by loss of radioactivity from 25-hydroxy[23,24-³H]vitamin D₃ and 25-hydroxy-[26,27-methyl-³H]vitamin D₃, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D₃, and structurally characterized as 23,25,26-trihydroxyvitamin D₃. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH)₃D₃ or 25(OH)D₃ over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.     

Production in vitro of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone from 1 alpha,25-dihydroxyvitamin D2 by rat small intestinal mucosa homogenates

The metabolism of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] in the rat has been studied under both in vivo and in vitro conditions. A time course study of the appearance of 1α,25-dihydroxyvitamin D₃-26,23-lactone in the plasma following intravenous or oral administration of 1α,25(OH)₂D₃ suggests that the small intestine may take part in production of the 1α,25(OH)₂D₃-26,23-lactone. In an in vitro study using a homogenate of rat small intestinal mucosa, 1α,25(OH)₂D₃ undergoes further metabolism to give more polar metabolite(s) which comigrate with authentic 1α,24,25-trihydroxyvitamin D₃ [1α,24,25(OH)₃D₃] on Sephadex LH-20 column chromatography. The metabolic profile obtained after high-pressure liquid chromatography reveals two major classes of metabolites, designated Peaks X and Y. Peak X is an unidentified metabolite of 1α,25(OH)₂D₃. Peak Y is chromatographically identical with 1α,25-dihydroxyvitamin D₃-26,23-lactone which has been recently isolated from the plasma of rats and dogs as a major metabolite produced in vivo from either 1α,25(OH)₂D₃ or 1α-hydroxyvitamin D₃ (N. Ohnuma, K. Bannai, H. Yamaguchi, Y. Hashimoto, and A. W. Norman, 1980, Arch. Biochem. Biophys.204, 387). The enzyme activity which produces metabolites X and Y in the rat intestinal homogenates is induced in vitamin D-replete rats by pretreatment of the animals with intravenous 1.25 μg/kg doses of 1α,25-dihydroxyvitamin D₃, 6 to 8 h previously.     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.Our findings suggest the vitamin D₃ effect is mediated locally.Key Words: kidney, organogenesis, transplantation     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.

Our findings suggest the vitamin D₃ effect is mediated locally.     

The association of 25-hydroxyvitamin D3 and D2 with behavioural problems in childhood

Background

Higher serum concentrations of 25-hydroxyvitamin D (25(OH)D), an indicator of vitamin D synthesis and intake, have been associated with better mental health and cognitive function. Concentrations of 1,25-dihydroxyvitamin D₃ (the active vitamin D₃ metabolite) have been associated with openness and extrovert behaviour, but 25(OH)D concentrations have not been associated with behavioural problems in humans.

Methods

We investigated the prospective association between the different forms of 25(OH)D - 25(OH)D₃ and 25(OH)D₂– and childhood behavioural problems in Avon Longitudinal Study of Parents and Children (ALSPAC). Serum 25(OH)D₃ and 25(OH)D₂ concentrations were assessed at mean age 9.9 years. Incident behavioural problems were assessed with Strengths and Difficulties Questionnaire (SDQ; emotional symptoms, conduct problems, hyperactivity-inattention problems, peer relationship problems and pro-social behaviour subscales and total difficulties score) at mean age 11.7. Sample sizes varied between 2413-2666 depending on the outcome.

Results

Higher 25(OH)D₃ concentrations were weakly associated with lower risk of prosocial problems (fully adjusted odds ratio: OR (95% confidence interval: CI) 0.85 (0.74, 0.98)). Serum 25(OH)D₃ or 25(OH)D₂ concentrations were not associated with other subscales of SDQ or total difficulties score after adjusting for concfounders and other measured analytes related to vitamin D.

Conclusions

Our findings do not support the hypothesis that 25-hydroxyvitamin D status in childhood has important influences on behavioural traits in humans.     

Effect of vitamin D treatments on plasma metabolism and immune parameters of healthy dairy cows

The objective of this study was to investigate the possible beneficial effect of vitamin D repletion on certain immune parameters of vitamin D insufficient dairy cows. Twenty dairy cows in late lactation were treated daily with vitamin D in five different ways: sunlight exposure (SUN), D₂ supplementation combined with sunlight exposure (D2SUN), D₂ supplementation (D2), D₃ supplementation (D3), and D₂ and D₃ supplementation combined (D2D3). The cows had very low vitamin D levels at d 0 because of the vitamin D deprivation before the study. After 1 month of vitamin D repletion, all cows had plasma 25(OH)D levels within the normal range. Total 25(OH)D concentration was significantly higher in SUN, D2SUN and D2D3 than D2 or D3 at the end of the study. However, milk yield, as well as protein and fat content of the milk, was not influenced by vitamin D treatments. There was no difference obtained in the measured immune parameters: Leucocyte populations, somatic cell count, immunoglobulin concentrations in plasma and milk, and antigen-stimulated cytokine productions did not change in response to vitamin D repletion or difference in vitamin D sources, and no relations to plasma 25(OH)D levels were identified. Despite the fact that plasma 25(OH)D increased from a very low level to normal range, the present study did not show any effect of vitamin D repletion on the tested immune parameters of healthy dairy cows. Therefore, in this study, it was concluded that repletion to physiologically normal plasma 25-hydroxyvitamin D levels of vitamin D-depleted healthy dairy cows had no influence on immune parameters.     

Vitamin D in adipose tissue and serum 25-hydroxyvitamin D after roux-en-Y gastric bypass

Vitamin D is stored in body fat. The purpose of this study was to determine vitamin D concentration in abdominal fat of obese patients who underwent roux‐en‐Y gastric bypass (RYGB), and to describe changes in serum 25‐hydroxyvitamin D (25(OH)D) levels in relation to loss of body fat. Subjects from a single clinic who were scheduled for RYGB were invited into the study. Abdominal subcutaneous, omental, and mesenteric fat were obtained at time of surgery. Adipose vitamin D₂ and vitamin D₃ concentrations were measured by high‐performance liquid chromatography (HPLC). Weight and serum 25(OH)D were assessed at baseline and every 3 months up to 1 year. Seventeen subjects were included, and fat samples were available from eleven. Total vitamin D content in subcutaneous abdominal fat was 297.2 ± 727.7 ng/g tissue, and a wide range was observed (4–2,470 ng/g). Both vitamin D₂ and vitamin D₃ were detected in some of the fat samples. At baseline, 25(OH)D was 23.1 ± 12.6 ng/ml. Average weight loss was 54.8 kg at 12 months, of which ～40 kg was fat mass. Despite daily vitamin D intake of ≥2,500 IU throughout the study, no significant increase in serum 25(OH)D was observed, with mean serum concentration of 25(OH)D at 1 year of 26.2 ± 5.36 ng/ml (P = 0.58). We conclude that vitamin D in adipose tissue does not significantly contribute to serum 25(OH)D despite dramatic loss of fat mass after RYGB.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃