Phosphate transport by embryonic chick duodenum. Stimulation by vitamin D3.

Embryonic chick duodenum maintained in organ culture is a well-suited model for the study of vitamin D effects on inorganic phosphate (Pi) absorption. The system is sensitive to as little as 6.5 nM vitamin D3 (0.1.I.U./ml culture medium). Increased phosphate absorption is observed after 6--12 h of culture. Maximal response (133% of vitamin D-efficient control) is achieved at 24 h. Phosphate uptake by embryonic chick duodenum involves a saturable and a non-saturable component. The former displays characteristics of an active sodium-dependent transport mechanism and is also sensitive to vitamin D3. Presence of the sterol in culture medium raises the maximal velocity from 55 to 75 nmol Pi/min per g tissue. Km remains unchanged (0.5 mM Pi). Duodena cultured in presence of inhibitors of protein synthesis (actinomycin D, alpha-amanitin and cycloheximide) display reduced rates of phosphate absorption. This treatment also prevents vitamin D3 action on phosphate transport. It is concluded that the sterol affects phosphate transport by modulation of synthesis of proteins which are functional in the Pi absorptive process.     

The effect of vitamin D3 metabolites on normal and leukemic bone marrow cells in vitro

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.     

Structural changes in parathyroid glands exposed to vitamin D metabolites in vitro

Isolated parathyroid glands from normal adult Mongolian gerbils were incubated for 10 minutes to 16 1/2 hours at high, medium or low concentrations of Ca2+, with or without added 1,25-dihydroxycholecalciferol (1.25-DHCC) or 25-hydroxycholecalciferol (25-HCC), after which they were studied electron microscopically. 1,25-DHCC appeared to potentiate the inhibitory action of high calcium on parathyroid activity, whereas 25-HCC had no structurally detectable effect.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

The influence of substitution at C24 on the calcium-binding protein-stimulating activity of vitamin D metabolites in chick embryonic duodenum

The production of calcium-binding protein, in vitro, by embryonic chick duodenum has been used to assess the potency of vitamin D compounds. The introduction of an hydroxyl on 1-, 25-, or 24R-position enhanced biological activity while the introduction of both 1α- and 25-hydroxyls produced maximal activity. However 24R-hydroxylation of 1,25-dihydroxyvitamin D₃ diminished activity. The vitamin D₂ side chain on 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D did not greatly diminish activity in contrast to the fact that the vitamin D₂ compounds are 10% as active as the vitamin D₃ compounds in vivo in the chick. These results support the idea that the target organs of the chick do not discriminate against the vitamin D₂ side chain and that the discrimination in this species is at the level of metabolism.     

Renal parathyroid hormone-dependent adenylate cyclase in vitamin D-deficient rats. Inhibition by hydroxylated vitamin D3 metabolites

The adenylate cyclase activation by bovine synthetic parathyroid hormone (bPTH) (1-34) was studied in vitro in kidney plasma membranes from D-deficient (D-Mb) or normal (D+Mb) rats. In D-Mb, the apparent affinity of parathyroid hormone (PTH) for membranes (170 +/- 30 nM) was significantly higher than that measured in D+Mb (55 +/- 5 nM). The maximum velocity of the PTH-stimulated adenylate cyclase was significantly higher in D+Mb than in D-Mb (163.0 +/- 13.7 and 93.4 +/- 6.7 pmol of cAMP/mg of protein/min, respectively). The action of vitamin D metabolites on the adenylate cyclase stimulation by PTH was then studied in vitro in D-Mb and D+Mb. In D-Mb, 25-hydroxyvitamin D3, 24,25-, and 1, 25-dihydroxyvitamin D3 significantly inhibited cAMP production in the presence of 0.87 microM of bPTH. Vitamin D3 had no effect. Maximal inhibition (86%) was observed for 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 decreased the maximum velocity of PTH-stimulated adenylate cyclase but did not modify the bPTH apparent affinity for D-Mb. The vitamin D3 metabolites tested did not modify the cyclase stimulation by isoproterenol, sodium fluoride, or 5'-guanylylimidodiphosphate. The presence of 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 did not increase the (Na-K)-ATPase or the phosphodiesterase activities. In the presence of 1,25-dihydroxyvitamin D3 and bPTH, the apparent affinity of ATP for the catalytic moiety was not modified. The maximum velocity was decreased. These results suggest an in vitro interaction between hydroxylated vitamin D metabolites and kidney membranes PTH receptor.     

The effect of inhibitors of protein and RNA synthesis on 1 alpha,25-dihydroxyvitamin D3-dependent calcium uptake in cultured embryonic chick duodenum

To determine whether 1 alpha, 25-dihydroxyvitamin D3-dependent increases in intestinal calcium uptake require de novo protein and RNA synthesis, the effects of several inhibitors of these processes have been re-examined in vitro using cultured embryonic chick duodenum. To minimize the contributions of antibiotic toxicity to the interpretation of results, care was taken to examine inhibitor effects at early times after the onset of the 1 alpha, 25-dihydroxyvitamin D3 response. Cycloheximide at a concentration of 5 microM blocked hormone-dependent calcium uptake at all times examined (6 to 24 h). Actinomycin D was similarly effective at 6 to 12 h. The effects of cycloheximide were totally reversible while actinomycin D inhibition was only partially reversible. These compounds inhibited protein or RNA synthesis by 68.4 +/- 1.4 and 51.4 +/- 1.1%, respectively. Anisomycin, another inhibitor of polypeptide chain elongation and alpha-amanitin, an inhibitor of RNA polymerase I, also blocked 1 alpha, 25-dihydroxyvitamin D3-dependent calcium uptake after 12 h in culture. These results further strengthen the hypothesis that 1 alpha, 25-dihydroxyvitamin D3 stimulates intestinal calcium transport via a nuclear mechanism involving new gene expression.     

Effect of culture conditions on the synthesis of vitamin D(3) metabolites in Solanum glaucophyllum grown in vitro

In cultured Solanum glaucophyllum we have recently described the operation of a light-independent pathway of 1alpha,25-dihydroxy-vitamin D(3) (1alpha,25(OH)(2)D(3)) biosynthesis which involves similar intermediates as in vertebrates. In this work we investigated factors influencing the formation of 1alpha,25(OH)(2)D(3) and related sterols in S. glaucophyllum grown in vitro in darkness. Callus tissue and cells cultured in Murashige and Skoog medium in the absence of light were employed. Lipids were extracted with chloroform-methanol. The remaining water soluble fraction was incubated with beta-glucosidase to release vitamin D(3) compounds from their glycoconjugated derivatives followed by organic solvent extraction. Vitamin D(3) derivatives were isolated by Sephadex LH-20 and high-performance liquid chromatography (HPLC). HPLC or competitive protein binding assays with intestine 1alpha,25(OH)(2)D(3) receptor and serum vitamin D binding protein were used to quantify the metabolites. The levels of 1alpha,25(OH)(2)D(3) in calli varied according to the tissue explant origin, e.g. stem>leaf>fruit. For all organs, the metabolite was mainly present as free sterol (>80% of total). There were larger amounts of 25(OH)D(3) than 1alpha,25(OH)(2)D(3). It was found that Ca(2+), auxin and kinetin are important factors upregulating 1alpha,25(OH)(2)D(3) synthesis in S. glaucophyllum tissue and cells. Irradiation with UV light increased vitamin D(3) but not 1alpha,25(OH)(2)D(3) levels. In agreement with these results, incubation of cells with [3H]25(OH)D(3) revealed a low conversion rate to [3H]1alpha,25(OH)(2)D(3). The operation of a light-dependent pathway formation of vitamin D(3) coupled to higher expression of 25(OH)D(3)-1alpha-hydroxylase may account for the large concentrations of 1alpha,25(OH)(2)D(3) normally found in differentiated field-grown plants.     

Impaired mineral metabolism in Cushing's syndrome: parathyroid function, vitamin D metabolites and osteopenia

To clarify the mechanism for the impaired mineral metabolism in Cushing's syndrome, the clinical features, biochemical parameters before and after oral calcium load, and vitamin D metabolism were compared between two groups of patients of endogenous Cushing's syndrome (17 cases) with and without osteopenia. The patients with osteopenia [OP (+): 7 cases, all female] were older (42.7 +/- 8.3 y. o.) and had a longer duration (117 +/- 75 M) of the syndrome than those without osteopenia [OP (-): 33.8 +/- 8.9 y. o., 36 +/- 25 M]. OP (-) showed a blunted hypercalciuria after oral calcium load (63.7 +/- 20.4 to 90.9 +/- 36.1 mg/g X Cr), while OP (+) had higher levels of urinary excretion of calcium (fasting: 120.4 +/- 37.5, and after oral calcium load: 235.6 +/- 72.6 mg/g X Cr), of cyclic AMP (7.6 +/- 1.1 nmol/dl X GF), and of plasma 1.25(OH)2D (76.6 +/- 34.0 pg/ml) than OP (-) (u-cAMP: 3.2 +/- 2.0 nmol/dl X GF, 1,25(OH)2D: 27.9 +/- 16.3 pg/ml). These results indicate that 1) elderly female patients with Cushing's syndrome of long duration are susceptible to OP, 2) during the early phases of the syndrome, reduced intestinal calcium absorption with sustained calciuria (probably through the inhibition of calcium reabsorptive effect of PTH by glucocorticoid) induces negative calcium balance, leading to 3) a development of secondary hyperparathyroidism which stimulates 1,25(OH)2D synthesis. Thus, the mechanism involving bone resorption stimulated by excess PTH along with the direct inhibition of bone formation by glucocorticoid seems to play an important role in a progressive development of OP in Cushing's syndrome.     

The biological assessment of vitamin D3 metabolites produced by rumen bacteria

Biological assays were performed to evaluate 10-oxo-19-nor-vitamin D3 (10-oxo-D3) and 5(E) 25-hydroxy-10-oxo-19-nor-vitamin D3 (25-OH-10-oxo-D3) two bacterial products of vitamin D3 (D3) and 25-hydroxyvitamin D3 (25-OHD3) metabolism, respectively. The 5(Z) and 5(E) isomers of 10-oxo-D3 were, respectively, 40- and 80-fold less active than D3 in stimulating Ca+2 absorption from the gut. 25-Hydroxy-10-oxo-D3 did not stimulate Ca+2 absorption. Only 5(Z) 10-oxo-D3 induced mobilization of bone Ca+2. In addition, both 10-oxo-D3 and 25-OH-10-oxo-D3 showed poor affinities for either the plasma D3-binding protein or the thymus 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor. 10-Keto-D3 exhibited a plasma half-life of only 6 min. This was a much shorter half-life than that exhibited by other vitamin D metabolites and was expected because of the poor affinity 10-oxo-D3 has for the plasma vitamin D binding protein. Bacterial metabolism of D3 deactivates the vitamin, which allows ruminants to tolerate relatively large oral doses of D3.     

The effect of vitamin D3 metabolites on membrane proteins of chick duodenal brush borders.

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.     

Seasonal variation and correlation analysis of vitamin D and parathyroid hormone in Hangzhou,Southeast China

This study aimed to describe the 25‐hydroxyvitamin D (25(OH)D) and parathyroid hormone (PTH) status of Southeast Chinese individuals influenced by season. The secondary aim was to determine the cutoff for sufficient 25(OH)D in a four‐season region. From January 2011 to June 2014, a total of 17 646 individuals were evaluated in our study. The serum levels of PTH were detected simultaneously in 5579 cases. A total of 25(OH)D and intact PTH were measured by the electrochemiluminescent immunoassay. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D and PTH were studied. The mean 25(OH)D concentration in our study was 43.00(30.40) nmol/L. The prevalence of insufficiency (25(OH)D < 50 nmol/L) was 62.87% and that of deficiency (<30 nmol/L) was 28.54%. Mean serum 25(OH)D levels revealed a limited sinusoidal profile throughout the year and were significantly higher in Autumn. On the other hand, PTH levels showed an opposite response to seasonal effects relative to 25(OH)D. Age, BMI and daylight were not significantly correlated with 25(OH)D and serum PTH reached a plateau at higher values of serum 25(OH)D of 42.86 nmol/L. This study demonstrated that Vitamin D insufficiency is highly prevalent in Southeast China. The concentration of 25(OH)D in the male group was generally higher than that in the female group. Seasonal variation was an important aspect of 25(OH)D and PTH concentration. This study revealed that the optimal serum threshold of 25(OH)D for bone health should be between 40 and 50 nmol/L for Southeast Chinese individuals.     

The quantitative determination of vitamin D3 and its metabolites in plasma

A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease.