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D. Faict, P. De Moor, R. Bouillon, W. Heyns, H.-J. Heiniger, D. Corrow, and E. Lesaffre

ABSTRACT

The influence of age, sex and strain on the serum concentration of transcortin (corticosteroid-binding globulin) and vitamin D-binding protein (DBP) in mice was investigated. The effect of age was studied in two strains, C57BL/6JPfd and BALB/cmHeAPfd. The concentration of transcortin and DBP increased with age. In young animals the concentration of each protein showed a significant strain difference, which disappeared in older mice for DBP, but not for transcortin. In 7-day-old animals, no sex difference was observed for either protein, but in older animals a clear sex difference was found for transcortin. Adult males tended to have somewhat higher levels of DBP than adult females, but this difference was significant only on day 70.

The variation in transcortin and DBP levels was further investigated in a large number of mouse strains. The DBP concentration did not markedly vary among strains (5·98–9·65 μmol/l in males and 5·08–8·85 μmol/l in females). Transcortin, however, showed marked strain variations, ranging from 0·72 to 2·06 μmol/l in males and from 1·02 to 4·55 μmol/l in females and there was a significant correlation (r= 0·66, n= 26, P<0·001) between the mean transcortin levels in males and females of different strains. Interstrain variation was much higher than intrastrain variation or variation among related strains, suggesting that the transcortin concentration is largely controlled by genetically determined factors.

There was a significant correlation (r= 0·82, n = 9, P<0·01) between the mean corticosterone and transcortin concentrations (measured at 21.00 h). Consequently, differences in the free corticosterone levels in the serum of various mouse strains were smaller than the differences in the total corticosterone concentrations. The affinity of transcortin for corticosterone was similar in all but one strain; however, transcortin of the RIIIS/J strain showed a lower affinity for corticosterone.

J. Endocr. (1986) 109, 141–147

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S. K. Abbas, A. D. Care, H. Van Baelen, and R. Bouillon

ABSTRACT

A radioimmunoassay for ovine vitamin D-binding protein (DBP) has been developed. This assay can also effectively measure DBP in goat plasma. A suitable ovine DBP antiserum raised in a rabbit produced a single monospecific line of precipitation when reacted against purified sheep DBP and sheep plasma. The preliminary purification of 125I-labelled ovine DBP was carried out using adsorption chromatography, and the final purification immediately before addition to the assay tubes was achieved by high-pressure liquid chromatography. Displacement of 125I-labelled ovine DBP by dilutions of sheep and goat plasma or standard DBP gave parallel curves, and only weak competition was observed with calf and pig plasma. The assay detected as little as 26 pmol DBP/1 with intra- and interassay coefficients of variation of 3 and 14% respectively. The mean plasma concentration of DBP in nine pregnant sheep (110–120 days of gestation) was 8·7 ± 0·3 (s.e.m.) μmol/l. These levels were significantly (P<0·02; paired t-test) higher than those in matched fetal plasma (6·7 ±0·4 μmol/l) obtained in utero through a catheter in a carotid artery. Plasma DBP concentrations in pregnant sheep were also significantly (P<0·02) higher than in five normal non-pregnant sheep (6·8 ± 0·5 μmol/l). The mean concentrations of total 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in maternal and fetal plasma were 92·0 ±8·7 pmol/l and 152·5±18·0 pmol/l respectively (P<0·05). The free 1,25-(OH)2D3 index, a measure of the level of free 1,25-(OH)2D3 in plasma, was calculated as the ratio between the molar concentrations of total 1,25-(OH)2D3 and DBP. The mean value of the free 1,25-(OH)2D3 index in the fetus (2·3 × 10−5) was significantly (P<0·01) higher than that in the dam (1·1 × 10−5), thus indicating a gradient of free 1,25-(OH)2D3 during the latter part of ovine pregnancy with a mean fetal/maternal ratio of 2·1.

J. Endocr. (1987) 115, 7–12

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Ankana Ganguly, Jennifer A Tamblyn, Alexandra Shattock, Annsha Joseph, Dean P Larner, Carl Jenkinson, Janesh Gupta, Janesh R Gross, and Martin Hewison

Introduction Vitamin D binding protein (DBP) is a serum globulin associated with the systemic transport of vitamin D metabolites ( Chun 2012 ). Glomerular filtration of DBP and its primary cargo, the main circulating form of vitamin D, 25

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Y. Nys, R. Bouillon, H. Van Baelen, and J. Williams

ABSTRACT

The concentration of 25-hydroxyvitamin D3-binding protein (DBP) was measured, by immunodiffusion, in the blood of chickens from embryonic stages to sexual maturity. Low levels of DBP and 1,25-(OH)2D3 were detectable in the blood of chick embryos from the 12th and 17th day of incubation respectively and stayed at the same low levels until hatching. The blood concentration of DBP doubled between the 1st and 5th days of life, then increased slowly and reached the mean level of the adult male at 7–8 weeks of age. The concentration of DBP was independent of vitamin D status in growing chickens. A large increase was observed in DBP blood levels in hens just before sexual maturity. This change, and those observed in moulting hens, followed the variations in plasma concentrations of oestradiol more closely than those of progesterone or testosterone. Moreover, a large increase in plasma DBP levels was induced in immature chickens by oestradiol (0·5 mg/day), but not by testosterone or progesterone. Finally, the experimental suppression of egg shell formation and the associated decrease in 1,25-(OH)2D3 plasma levels had no effect on plasma DBP concentrations. However, 1,25-(OH)2D3 and DBP levels were higher in hens laying shell-less eggs than in immature pullets. The increases in DBP levels at hatching, in immature pullets treated with oestrogens, in hens laying uncalcified eggs and at the onset of egg production were associated with increases in 1,25-(OH)2D3, suggesting a relationship between the levels of DBP and 1,25-(OH)2D3 in the blood.

J. Endocr. (1986) 108, 81–87

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B. L. Nyomba, R. Bouillon, and P. De Moor

ABSTRACT

Vitamin D metabolites and vitamin D-binding protein (DBP) were measured in non-diabetic rats and in rats made diabetic with streptozotocin. The animals were studied in the intact state, after gonadectomy and during pregnancy. In male non-diabetic rats the serum concentrations of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and DBP decreased after orchidectomy and were restored by treatment with testosterone. In female non-diabetic rats, these parameters increased after ovariectomy. Increased 1,25-(OH)2D3 and decreased DBP concentrations were found during pregnancy in non-diabetic rats.

After the induction of diabetes in intact rats of both sexes, the concentration of DBP decreased, but a significant decrease in the concentration of 1,25-(OH)2D3 was found in male animals only. After ovariectomy, however, 1,25-(OH)2D3 decreased also in female diabetic rats.

Both orchidectomy and insulin deficiency depressed serum concentrations of 1,25-(OH)2D3 (−22 and −45% respectively) and DBP (−14 and −29% respectively), but the effects of insulin deficiency were greater than those of androgen withdrawal. Moreover, the testosterone concentration was twofold lower in intact male diabetic rats than in non-diabetic animals. Insulin, but not testosterone treatment, however, restored DBP and 1,25-(OH)2D3 concentrations in diabetic rats, and insulin was effective in intact as well as in gonadectomized animals.

This study shows that insulin deficiency decreases the concentrations of DBP and 1,25-(OH)2D3 in the rat, and that these decreases are facilitated by androgens, but counteracted by oestrogens.

J. Endocr. (1987) 115, 295–301

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C Farquharson, J S Rennie, N Loveridge, and C C Whitehead

Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is regarded as the most biologically active metabolite of cholecalciferol. It prevents tibial dyschondroplasia (TD) in chicks where inhibition of chondrocyte differentiation within the growth plate occurs. However, it is unclear whether its mode of action is through direct interaction with its chondrocyte receptor and its known regulatory role in cell differentiation or is mediated by increased calcium absorption and mobilisation. Synthetic analogues of 1,25(OH)2D3 such as 1,25-dihydroxy-16-ene-23-yne cholecalciferol (RO 23–7553) with increased differentiation properties but reduced calcaemic activity have been synthesised. In this study, the in vitro and in vivo effects of 1,25(OH)2D3 and RO 23–7553 on chick chondrocyte growth and differentiation were examined. In addition, the in vivo effectiveness of these steroids in preventing TD in chicks was assessed. 1,25(OH)2D3 and RO 23–7553 (10−12-10−7 m) displayed biphasic concentration effects and had similar potencies in vitro in regulating chondrocyte proliferation and differentiation. However, while the incidence of TD in birds dosed with 1,25(OH)2D3 was lower (10%) than in control chicks (55%), RO 23–7553 was ineffective (50%). This may be the result of its reduced affinity (1000 times less) for the plasma vitamin D binding protein (DBP) and the chondrocyte receptor in comparison to that of 1,25(OH)2D3. A reduction in calcium supply to the chondrocyte may also result in decreased chondrocyte differentiation but blood ionised and plasma total calcium were normal in birds dosed with RO 23–7553. These data suggest that RO 23–7553 and 1,25(OH)2D3 regulate chondrocyte proliferation and differentiation similarly in vitro but not in vivo. This may be caused by differences in DBP binding and clearance rates of the two steroids in vivo.

Journal of Endocrinology (1996) 148, 465–474

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I Nemere, D Yazzie-Atkinson, DO Johns, and D Larsson

An earlier study revealed that 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)D(3)) inhibits the rapid actions of 1,25(OH)(2)D(3) on stimulation of calcium transport in perfused duodena, as well as activation of protein kinases C and A. In the present work, a specific binding protein (24,25-BP) has been identified and partially characterized. Percoll-gradient resolution of differential centrifugation fractions from mucosal homogenates revealed the highest levels of specific [(3)H]24R,25(OH)(2)D(3) binding to be in lysosomes (approximately eight to tenfold greater than in basal lateral membrane fractions). Incubation of isolated enterocytes with 6.5 nM [(3)H]24R,25(OH)(2)D(3) for 10 s also demonstrated targeting of the steroid to lysosomal fractions. Using freshly isolated lysosomal fractions, time course studies indicated maximal specific binding after a 2-h incubation on ice. Western analyses revealed that the serum transport protein, DBP (vitamin D binding protein), was absent from both lysosomal and basal lateral membrane fractions. Protein dependence studies demonstrated linear binding between 0.05 and 0.155 mg of lysosomal protein. Saturation analyses yielded K(d)=7.4+/- 1.8 nM, B(max)=142+/-16 fmol/mg protein for lysosomes, and K(d)=8.5 nM, B(max)=149+/-25 fmol/mg protein for basal lateral membranes. Hill analyses of lysosomal binding yielded a Hill coefficient of 0.57+/-0.11, indicative of negative cooperativity. Studies with lysosomal proteins revealed a 81%+/-7% competition of 24S,25(OH)(2)D(3) with [(3)H]24R,25(OH)(2)D(3) for binding (P>0.05, relative to competition with 24R,25(OH)(2)D(3)), while 25(OH)D(3) and 1,25(OH)(2)D(3) yielded 53%+/-13% and 39%+/-11% competition respectively (each, P<0.05, relative to competition with 24R,25(OH)(2)D(3)). The apparent affinity of 24S,25(OH)(2)D(3) for 24,25-BP led to testing of the metabolites effectiveness in the perfused duodenal loop system. Vascular perfusion with 130 pM 1,25(OH)(2)D(3) stimulated (45)Ca transport to 2.5-fold above control levels after 40 min, while simultaneous perfusion with 6.5 nM 24S,25(OH)(2)D(3) and 130 pM 1,25(OH)(2)D(3) abolished the stimulatory activity completely. Purification of the 24,25-BP by chromatography revealed a single protein band upon SDS-PAGE and silver staining of 66 kDa. The combined results suggest that 24R,25(OH)(2)D(3) may mediate its hormonal activities through a specific binding protein.

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S. MUDHER, I. P. BRAIDMAN, M. HALES, and D. C. ANDERSON

The binding of 1,25-dihydroxy[3H]cholecalciferol (1,25(OH)2[3H]D3) and 25-hydroxy-[3H]cholecalciferol (25(OH)[3H]D3) in vitamin D target and non-target tissues from the fetal rat has been compared using two incubation conditions, each followed by charcoal adsorption and sucrose gradient centrifugation. In intact tissue incubations, equilibrium with the ligand was established overnight at 4 °C before cell disruption. In pre-prepared cytosol incubations, cytosol was prepared from the tissue before incubation with ligand. With both ligands, more sterol was bound during pre-prepared cytosol incubation despite the use of fourfold lower ligand concentrations. With 1,25(OH)2[3H]D3, intact tissue incubation led to most marked binding with calvaria, which was preferentially displaced by unlabelled 1,25(OH)2D3; radioactivity sedimented almost entirely as a 3·2–3·7S peak (peak I) which was completely displaced by 100-fold excess 1,25(OH)2D3 but only partially by 25(OH)D3. Fetal small intestine, another putative target tissue, also showed displaceable binding of 1,25(OH)2D3; however, this was much less marked, and was not accompanied by a significant peak on sucrose gradients. Other fetal tissues (large intestine, kidney, skin, brain and heart) did not show significant displaceable binding of 1,25(OH)2D3 in intact tissue. In contrast, when intact calvaria, kidney, brain and heart were incubated with 25(OH)D3 they all showed significant displaceable binding to a 5·0–5·7S peak (peak II).

Incubations with pre-prepared cytosol confirmed ubiquitous binding of both sterols to peak II with the notable exception of that from small intestine; this peak II binding was preferential for 25(OH)D3, and is believed to be to the plasma vitamin D-binding protein (DBP). Only calvaria showed an additional peak I under these conditions, and even here it was dwarfed by peak II. We conclude that, at least in fetal bone, intact tissue incubations selectively detect receptor (peak I) binding of 1,25(OH)2D3 in the presence of the DBP. The substantial increase in binding of both sterols to peak II in pre-prepared cytosol compared with that in the same fetal tissues incubated intact suggests that some DBP is intracellular and therefore relatively inaccessible to extracellular sterol. The apparent absence of both peak I and peak II binding of cytosol from fetal small intestine merits further investigation.

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Rene F Chun, John S Adams, and Martin Hewison

vitamin D during macrophage innate immune responses. Serum 25-hydroxyvitamin D (25D) bound to vitamin D-binding protein (DBP) is internalized by macrophages either through passive diffusion of ‘free’ 25D or via megalin (meg)-mediated uptake. Intracellular

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Lauriane Bonnet, Esma Karkeni, Charlène Couturier, Julien Astier, Catherine Defoort, Ljubica Svilar, Franck Tourniaire, Lourdes Mounien, and Jean-François Landrier

serum and its regulation by albumin and the vitamin D-binding protein . Journal of Clinical Endocrinology and Metabolism 63 954 – 9 59 . ( https://doi.org/10.1210/jcem-63-4-954 ) Bikle D Bouillon R Thadhani R Schoenmakers I 2017 Vitamin