Hugo Van Baelen

Influence of the Vitamin D-binding Protein on the Serum Concentration of 1,25-Dihydroxyvitamin D3: SIGNIFICANCE OF THE FREE 1,25-DIHYDROXYVITAMIN D3 CONCENTRATION

The influence of the serum binding protein (DBP) for vitamin D and its metabolites on the concentration of its main ligands, 25-hydroxyvitamin D(3) (25-OHD(3)) and 1,25-dihydroxyvitamin D(3) (1,25-[OH](2)D(3)) was studied. The concentration of both 1,25-(OH)(2)D(3) and DBP in normal female subjects (45+/-14 ng/liter and 333+/-58 mg/liter, mean+/-SD, respectively; n = 58) increased during the intake of estro-progestogens (69+/-27 ng/liter and 488+/-90 mg/liter, respectively; n = 29), whereas the 25-OHD(3) concentration remained unchanged. A positive correlation was found between the concentrations of 1,25-(OH)(2)D(3) and DBP in these women. At the end of pregnancy, the total concentrations of 1,25-(OH)(2)D(3) (97+/-26 ng/liter, n = 40) and DBP (616+/-84 mg/liter) are both significantly higher than in nonpregnant females and paired cord serum samples (48+/-11 ng/liter and 266+/-41 mg/liter, respectively). A marked seasonal variation of 25-OHD(3) was observed in pregnant females and their infants, whereas in the same samples the concentrations of both DBP and 1,25-(OH)(2)D(3) remained constant throughout the year. The free 1,25-(OH)(2)D(3) index, calculated as the molar ratio of this steroid and DBP, remains normal in women taking estro-progestogens, however, and this might explain their normal intestinal calcium absorption despite a high total 1,25-(OH)(2)D(3) concentration. In pregnancy the free 1,25-(OH)(2)D(3) index remains normal up to 35 wk of gestation, but during the last weeks of gestation, the free 1,25-(OH)(2)D(3) index increases in both circulations. A highly significant correlation exists between the (total and free) 25-OHD(3) and 1,25-(OH)(2)D(3) concentrations in maternal and cord serum both at 35 and 40 wk of gestation.

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushings syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.

A structural basis for the unique binding features of the human vitamin D-binding protein.

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 Å crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.

Comparative study of the affinity of the serum vitamin d-binding protein

Abstract The affinity and capacity of the serum vitamin D-binding protein for 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 were studied. The association constant (M −1 ) at 4°C for 25-hydroxyvitamin D 3 was about 5 × 10 8 at pH 7.4 in all species (man, monkey, rat and chick) except for a 10-fold higher affinity in the toad. At pH 8.6 a tenfold increase in affinity for 25-hydroxyvitamin D 3 was observed in the mammalian species but not in the chick or the toad. The association constant (M −1 ) of the serum vitamin D-binding protein for 1,25-dihydroxyvitamin D 3 at 4°C and pH 7.4 was about 1.5 × 10 7 in man, monkey and chick and was tenfold higher in rat and toad. At pH 8.6 only a slight increase in affinity for 1,25-dihydroxyvitamin D 3 was observed. The serum binding capacity for 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 was in the micromolar order in all species except for a 100-fold lower capacity in the toad. The genetic heterogeneity of the human and rat vitamin D-binding protein did not affect its affinity or capacity for both vitamin D metabolites.

The Biological Activity of Nonsteroidal Vitamin D Hormone Analogs Lacking Both the C‐ and D‐Rings

1α,25‐dihydroxyvitamin D is a key calcium‐regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A‐ring and side chain, whereas relatively little is known about the requirements of the natural CD‐ring structure for the vitamin D–like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D‐region. This first series of nonsteroidal analogs consists of (1R,3S)‐5‐((Z,2E)‐4‐((1S,3S)‐3‐(4‐hydroxy‐4‐methylpentyl)‐1,2,2,‐trimethylcyclopentyl)‐2‐butenylidene)‐4‐methylenecyclohexane‐1,3‐diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C‐ and D‐rings and by the presence of an unnatural five‐membered ring which we call the E‐ring. KS 176 with the otherwise natural side chain structure of 1α,25(OH)2D3 has between 10 and 30% of the biological activity of 1α,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL‐60 and MG‐63; antiproliferating activity on MCF‐7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD‐rings are not mandatory for the biological activity of 1α,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A‐seco B‐rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.

Biological Activity of CD‐Ring Modified 1α,25‐Dihydroxyvitamin D Analogues: C‐Ring and Five‐Membered D‐Ring Analogues

Nonsteroidal analogues of 1α,25(OH)2D3, lacking either the full five‐membered D ring (C‐ring analogues) or the full six‐membered C ring (D‐ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1α,25(OH)2D3. Maximal superagonistic activity was seen for the C‐ring analogue with a 24(R)‐hydroxyl group in the side chain [30‐ to 60‐fold the activity of 1α,25(OH)2D3]. The 19‐nor‐16‐ene‐26,27‐bishomo C‐ring analogue showed the best ratio of antiproliferative to calcemic effects (1275‐fold better than 1α,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D‐dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR‐ligand complex more resistant against protease digestion than is 1α,25(OH)2D3. In contrast to 20‐epimer steroidal vitamin D analogues, 20‐epimer C‐ring analogues were less potent than analogues with a natural C‐20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1α,25(OH)2D3 despite lower receptor affinity and, for the C‐ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published. (J Bone Miner Res 2000;15;237–252)

Actin-DBP: the perfect structural fit?

The multifunctional vitamin D binding protein (DBP) is an actin-sequestering protein present in blood. The crystal structure of the actin-DBP complex was determined at 2.4 A resolution. DBP binds to actin subdomains 1 and 3 and occludes the cleft at the interface between these subdomains. Most remarkably, DBP demonstrates an unusually large actin-binding interface, far exceeding the binding-interface areas reported for other actin-binding proteins such as profilin, DNase I and gelsolin. The fast-growing side of actin monomers is blocked completely through a perfect structural fit with DBP, demonstrating how DBP effectively interferes with actin-filament formation. It establishes DBP as the hitherto best actin-sequestering protein and highlights its key role in suppressing and preventing extracellular actin polymerization.

Decreased cortisol-binding affinity of transcortin Leuven is associated with an amino acid substitution at residue-93

Genomic DNA was isolated from two related individuals who are homozygous for transcortin Leuven, a corticosteroid-binding globulin variant with decreased cortisol-binding affinity. This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the four exons that encode transcortin. Sequence analysis of these exons showed several mutations within the coding sequence of both individuals, but only one of these will result in an amino acid substitution. This mutation is located within exon 2 and alters the codon (CTC) normally associated with Leu-93 in the transcortin polypeptide to a codon (CAC) for histidine in the variant genes.

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

SummaryVitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)2D3, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated45Ca. We found that the resorptive response to 1,25(OH)2D3 was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)2D3 activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×107 M−1) than rDBP (5.9×107 M−1) for 1,25(OH)2D3 or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)2D3-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)2D3 is physiologically important.

The role of sialic acid in determining the survival of transcortin in the circulation

Abstract The effect of desialylation on the survival of human transcortin and of its ligand cortisol has been investigated using the isolated perfused rat liver preparation. In contrast with native transcortin, sialic acid-free transcortin was promptly cleared from the perfusate. The hepatic uptake was accompanied by a significant reduction of the cortisol half-life.