H. Van Baelen

Study of steroid-protein binding by means of competitive adsorption: Application to cortisol binding in plasma

The distribution of a steroid between a solution of steroid-binding proteins and a steroid-adsorbing solid added to this solution, depends not only on the properties of the adsorbent but also on the properties of the binding proteins and thus may be used for the study of steroid-protein interactions. Different types of distribution can be distinguished and were applied to the study of cortisol binding in plasma. 1. (1) Endogenous steroids were removed from plasma by incubation with a rather large amount of adsorbent. 2. (2) The cortisol-binding capacity of plasma transcortin was measured after incubation with a balanced amount of adsorbent and cortisol. 3. (3) A relative index of cortisol-protein binding proportional to the ratio of bound to unbound cortisol, was determined by incubation of charcoal-treated plasma with a trace of [1,2-3H]cortisol and appropriate amounts of adsorbent. 4. (4) From this relative index of cortisol—protein binding in plasma an estimate of the cortisol-binding affinity of plasma transcortin was derived.

Transport of vitamin D: significance of free and total concentrations of the vitamin D metabolites

Many hormones, nutrients, vitamins, and minerals are transported in the blood bound to specific carrier proteins. These ligand-protein interactions generally increase the solubility of the ligand in plasma, but it remains doubtful whether these proteins are required for the solubilization of the tiny final concentrations of some hormones and vitamins. One might, therefore , wonder which o ther functions have been the stimuli for the development of these transport proteins during the evolution of vertebrates. For several nutrients, vitamins, and minerals, the main function of their carrier protein is to facilitate the entry of the ligand in the specific target cells (Fig. 1A). In these cases the ligand-protein complex is first recognized and bound by cell membrane receptors, and the ligand or ligand-protein complex is thereafter internalized and incorporated into the cell metabolism. This nutrient type of carrier protein has been well de sc r ibed in r e cen t r ev iews on cholesterol [1] and vitamin A [2], and this model resembles more and more the fate of the polypeptide hormone-membrane receptor [3]. Deficiency or malfunctioning of this kind of carrier protein generally results in serious illness [1, 2]. The steroid and thyroid hormones are also bound to specific carrier proteins (Fig. 1B), but there is no experimental evidence of membrane receptors for either the ligand, carr ier protein, or ligand-carrier protein complex. On the contrary, the free or unbound hormone is believed to cross (freely?) the lipophilic cell membrane before binding to cytoplasmic and/or nuclear receptors. In contrast to the nutrient type model, the free and not the total hormone concentrat ion is thus available for cellular entry, and, in general, the free hormone levels correspond best to the physiological state. The evi-

Increases in calbindin D 28K mRNA in the uterus of the domestic fowl induced by sexual maturity and shell formation

Uterine concentrations of calbindin D 28K mRNA were measured in immature pullets and laying hens by dot-blot hybridization using a [32P]cRNA probe prepared from the calbindin cDNA. In immature pullets, estrogen increased the calbindin mRNA level and the plasma concentration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. When testosterone was administered with estrogen there was a further increase in calbindin and its mRNA and an increase in the free 1,25-(OH)2D3 index calculated as the ratio of the molar concentrations of total 1,25-(OH)2D3 and vitamin D-binding protein (DBP). In laying hens the uterine concentration of calbindin mRNA was low 4 hr after ovulation, but increased most markedly 12 and 18 hr later, when shell calcification took place. Calbindin concentration remained unchanged during the different stages of egg formation but was much higher in laying hens than in pullets treated with sex steroids. Suppression of shell formation by premature expulsion of the egg decreased the concentrations of calbindin mRNA and uterine calbindin and the free 1,25-(OH)2D3 index in the plasma. A concomitant increase in calbindin and its mRNA was observed at resumption of shell formation in hens previously laying shell-less eggs. Withdrawal of food for 44 hr decreased the uterine concentration of calbindin and its mRNA without a change in the free 1,25-(OH)2D3 index in the blood. It is concluded that the synthesis of uterine calbindin is stimulated primarily at sexual maturity and at calcification of the first shell by transcriptional processes. The daily increase in calbindin mRNA associated with shell formation and the absence of a concomitant change in calbindin concentration suggest that post-transcriptional processes exist and that stimuli other than the sex steroid or the 1,25-(OH)2D3 are involved in regulation of calbindin synthesis in the uterus.

Polyunsaturated fatty acids decrease the apparent affinity of vitamin D metabolites for human vitamin D-binding protein

The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D3 (25-OHD3) and 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD3 and 1,25-(OH)2D3 for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A1 and E1) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP. The apparent affinity of DBP for both 25-OHD3 and 1,25-(OH)2D3 decreased 2.4- to 4.6-fold in the presence of 36 microM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD3 to DBP by 20%. Much smaller ratios of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)2D3 to DBP. These latter ratios are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [3H]25-OHD3 to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids.

The effect of vitamin D analogs and of vitamin D-binding protein on lymphocyte proliferation

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.

Nonhypercalcemic vitamin D analogs: interactions with the vitamin D-binding protein.

The natural vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), not only regulates serum and bone calcium homeostasis but is probably also a paracrine factor in several cells and tissues including skin, immune system, placenta and brain, where it stimulates cell differentiation and inhibits cell proliferation. Several structural analogs of 1 alpha, 25-(OH)2D3 not only have superagonist activity but also display a selective action profile: indeed they maintain or have increased activity on cell differentiation/proliferation but also have substantially decreased calcemic activity when compared to 1 alpha,25-(OH)2D3. This decreased calcemic activity is partially due to mere pharmacological reasons: because of low binding affinity for the plasma vitamin D-binding protein, a more rapid extracellular metabolism and increased cellular uptake is possible when compared to 1 alpha,25-(OH)2D3. Their short extracellular half-life combined with comparable or enhanced transactivation potency together with analog-and cell-type-specific intracellular metabolism can probably explain why some analogs have a unique combination of superagonist activity and specific action profile with favorable dissociation of differentiation versus calcemic potency.

Regulation of calbindin D 28K and its mRNA in the intestine of the domestic hen

Intestinal calbindin synthesis in laying hens was analyzed to assess controlling factors operating during egg formation. In the absence of vitamin D, calbindin was not induced by estrogen and testosterone. In immature vitamin D-replete pullet, blood levels of 1,25(OH)2D3 increased in response to estrogen but the duodenal concentration of calbindin and its mRNA were increased only when testosterone was given together with estrogen. The plasma concentration of 1,25(OH)2D3 and the duodenal levels of calbindin and its mRNA were substantially higher in laying hens than in immature pullets. No differences in these parameters were observed between the stages of the ovulatory cycle. Suppression of shell formation for a week decreased the concentration of 1,25(OH)2D3 and of duodenal calbindin but did not affect the level of its mRNA. When egg shell formation resumed in hens previously laying shell-less eggs, the concentrations of 1,25(OH)2D3 and of calbindin and its mRNA increased toward the end of shell formation. A most important factor regulating intestinal calbindin synthesis in laying hens turned out to be 1,25(OH)2D3. Intestinal calbindin mRNA is more stable in laying hens than in young birds as its concentration declines more slowly when the stimulation provided by 1,25(OH)2D3 is withdrawn, as occurs following suppression of shell formation and after parathyroidectomy in laying birds. Intestinal calbindin mRNA is therefore increased by a process other than increasing 1,25(OH)2D3 formation. The factor influencing the stability of this mRNA in laying hens could be calcium. It is concluded that in hens the increased duodenal calbindin synthesis elicited by plasma 1,25(OH)2D3 at sexual maturity primarily involves a transcriptional process and the stabilization of the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological evaluation of 23-oxa-, 23-thia- and 23-oxa-24-oxo-1α,25-dihydroxyvitamin D3

Abstract The synthesis includes the side-chain construction starting from the Inhoffen-Lythgoe diol and coupling with the A ring. Both 23-oxa- and 23-thia-analogues showed a decreased cell differentiating effect but even a more decreased calcemic effect compared with 1α,25-(OH) 2 D 3 .

Plasma 1,25 dihydroxycholecalciferol and its free index are potentiated by ovulation dependent factors and shell formation induced hypocalcemia in the laying hens.

The time-course of the changes in blood ionized calcium, and in plasma 1,25 dihydroxycholecalciferol (1,25(OH)2D3) concentrations and its free index were studied in hens following suppression and resumption of shell formation and throughout the laying cycle in hens laying hard-shelled eggs, in hens fed a low or normal calcium diet and in hens laying shell-less eggs. The respective roles of the calcium needs for shell formation and of the reproductive status in regulation of 1,25(OH)2D3 production were analysed. Plasma 1,25(OH)2D3 decreased 3 hr after suppression of shell formation following premature egg expulsion and remained lower than that of hens laying hard-shelled eggs when premature expulsion of the eggs was continued for several days. Circulating 1,25(OH)2D3 tended to increase progressively when shell formation was resumed. Ablation of the parathyroid glands abolished this increase. In hens laying hard-shelled eggs, the plasma 1,25(OH)2D3 was higher during the period of shell secretion. Feeding hens a low calcium diet (1.2%) caused a marked increase in the plasma 1,25(OH)2D3. Ionized calcium levels tended to show reciprocal changes to plasma 1,25(OH)2D3 decreasing when calcification took place and increasing after its suppression. In hypercalcemic hens laying shell-less eggs and fed a 3.5% Ca diet, the plasma 1,25(OH)2D3 was at a high level 4 hr after ovulation and diminished thereafter. This additive stimulation does not, therefore, involve the parathyroid gland and may involve hormonal changes induced by ovulation. Vitamin D binding protein (DBP) in the plasma was at a high level in mature hens and was not affected by shell formation. Consequently, the free 1,25(OH)2D3 index fluctuated in parallel with total level of this hormone in mature hens. It is concluded that the calcium demand for shell formation modulates, in the short term, plasma 1,25(OH)2D3, via the homeostatic regulation of blood calcium by PTH, but that a large part of its increase is independent of PTH and is associated with the endocrine events concomitant with ovulation.

Synthesis and biological evaluation of some 25,26-epoxy-1α,24-dihydroxyvitamin D3 analogues

Abstract The synthesis of all stereoisomeric 25,26-epoxy-1α,24-dihydroxyvitamin D 3 analogues is described and relies on the Sharpless kinetic resolution of secondary alcohols. It further includes the Julia procedure for side chain construction and the Lythgoe A-ring coupling procedure. Biological evaluation includes the study of calcemic effect, receptor binding and cell differentiation. The synthesis and biological activities of vitamin D 3 analogues containing 25,26-epoxy function (X = OH or H) are presented.