Pharmacokinetic studies of vitamin D analogues: relationship to vitamin D binding protein (DBP)

Vitamin D(3), 25-hydroxyvitamin D(3) (25OHD(3)) and 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) bind to the vitamin D binding protein (DBP) in the serum. During the development of synthetic vitamin D analogues, it has been shown that the majority of analogues bind to DBP with a low affinity. This modifies their biological activitiesin vitro compared to 1α,25(OH)(2)D(3), since binding to DBP decreases the cellular uptake and access to the vitamin D receptor. It is therefore important to elucidate the possible role played by the binding or lack of binding to DBPin vivo. We have investigated the relationship between the binding affinity for human DBP and the serum level and serum half-life (t(1/2)) in rats of a series of new vitamin D analogues. The binding affinity for DBP was determined by displacement of(3)H-1,25(OH)(2)D(3) from DBP attached to Affi-Gel 10. The serum levels in rats following a single intravenous dose were assessed by HPLC and the serum half-life was determined for each analogue. In the group of vitamin D analogues which showed a low or no affinity for DBP, we have identified compounds with a short t(1/2) and compounds with a long t(1/2), all characterized by low initial serum levels. Compounds with a long t(1/2) were also found in the group with a high affinity for DBP, and they were easily identifiable by their high initial serum level. These results showed that the initial serum level of vitamin D analogues correlated with the affinity for DBP, but that there seemed to be no correlation with the metabolic rate as reflected by measurement of the serum half-life of the analogues.     

Distribution of Gc protein (vitamin D binding protein) on the surfaces of normal human lymphocytes and leukemic lymphocytes

Distribution of membrane-bound Gc protein (mGc) in normal lymphocyte subpopulations and leukemic lymphocytes was studied using a rabbit antiserum specific to Gc protein. Forty to fifty percent of the normal peripheral blood mononuclear cells were stained with anti-Gc antiserum by membrane immunofluorescence, and almost all of the B cells (B1+) and NK cells (Leu 11+) were mGc-positive. Only a few resting T cells (T3+) reacted with anti-Gc, but mGc appeared in a significant number of the T cells after activation by PHA. The distribution of mGc on leukemic lymphocytes of various types nearly corresponded to that in normal lymphocyte subpopulations.     

Identification and measurement of sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) in human saliva

Sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) were detected by specific immunoassays in mixed-saliva taken from normal men and women. The immunochemical identity of both proteins was demonstrated by parallelism between dose response curves generated by serial dilutions of saliva and standards in the immunoassays. In addition, the specific removal of both proteins by incubation with steroid affinity-chromatography gels demonstrated the integrity of their steroid binding activity. The mean +/- SD concentrations of SHBG (pmol/l) and CBG (microgram/l) in mixed-saliva taken from normal volunteers were as follows: men (n = 6) 19 +/- 10 and 38 +/- 18; women (n = 6) 63 +/- 60 and 72 +/- 71 and women during late pregnancy (n = 6) 282 +/- 168 and 92 +/- 80, respectively. The data indicate that the concentrations of SHBG and CBG in saliva represent approximately 0.1% of plasma concentrations. It is suggested that these proteins pass from the blood to saliva in a non-specific manner, and may influence the steroid content of saliva under certain physiological circumstances.     

Characterization of a monoclonal antibody to human serum vitamin D binding protein (Gc globulin): recognition of an epitope hidden in membranes of circulating monocytes

We have developed a murine hybridoma cell line that secretes a monoclonal antibody directed to the serum human vitamin D binding protein (hDBP), a 58,000-dalton alpha-globulin with a high avidity for 25-hydroxycholecalciferol and globular actin. This immunoglobulin G1 kappa-light chain antibody was produced by the fusion of the spleen cells from BALB/c mice, immunized with purified hDBP, with SP2/0-AG4 myeloma cells. The antibody was easily removed from the supernatant of hybridoma cultures or mouse ascites fluid by Protein A affinity chromatography. Apparent serum monospecificity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing gels transblotted to nylon membranes and overlayed with purified MAK 89 antibody and radioiodinated Protein A. The affinity of the antibody is high [dissociation constant (Kd) = 2.6 X 10(-11) M]. Parallel displacement of tracer by hDBP and human serum was observed. The sera from various species displaced the hDBP tracer in the following potency: monkey more than cat more than dog more than guinea pig. RIAs for DBP from several species are feasible with this antibody. This antibody does not, in contrast to polyclonal anti-hDBP antiserum, bind to viable monocytes. However, the MAK 89 antibody does bind to the membranes of well washed, fixed, and permeant circulating monocytes. Surface membrane radioiodination of monocytes and immunoprecipitation of the detergent lysates with the antibody demonstrates a protein with molecular weight equivalent to hDBP. The epitope recognized, therefore, appears to be hidden in the viable cells, suggesting an intimate and intricate association of the hDBP and monocyte plasma membrane.     

1,25-Dihydroxycholecalciferol inhibits the cochemotactic activity of Gc (vitamin D binding protein)

The identification of Gc (vitamin D binding protein) with the anionic polypeptide cochemotaxin has recently been reported. In this paper we investigate its dose dependent cochemotactic activity and report the inhibition of Gc enhanced chemotaxis by vitamin D3. These results further support the role of immunomodulating hormone played by vitamin D.     

维生素D结合蛋白研究进展

维生素D结合蛋白(DBP)最初被称为簇特异性成分(group-specific component),简称Gc蛋白,是一种多功能的糖蛋白,具有高度多态性,DBP基因多态性会影响其水平及其与维生素D及其代谢产物的亲和力,从而可能影响功能性25 (OH)D3水平.它不仅与骨质疏松相关,同时也与多种骨外疾病相关.     

Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [35S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated.     

Wolbachia surface protein (WSP) inhibits apoptosis in human neutrophils

Polymorphonuclear cells (PMNs) are essential for the innate immune response against invading bacteria. At the same time, modulation of PMNs' apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Wolbachia bacteria are Gram-negative endosymbionts of filarial nematodes and arthropods, phylogenetically related to the genera Anaplasma, Ehrlichia and Neorickettsia (family Anaplasmataceae). Although several pathogens are known to interfere with apoptosis, there is only limited information on specific proteins that modulate this phenomenon. This is the first evidence for the anti-apoptotic activity of a surface protein of Wolbachia from filarial nematode parasites (the Wolbachia surface protein, WSP). The inhibition of apoptosis was demonstrated on purified human PMNs in vitro by different methods. TUNEL assay showed that the percentage of dead cells was reduced after stimulation with WSP; Annexin V-FITC binding assay confirmed that cell death was due mainly to apoptosis and not to necrosis. Reduced caspase-3 activity in stimulated cells also confirmed an inhibition of the apoptotic process.     

A new ELISA method to identify the D327N mutation of human sex hormone binding globulin (SHBG)

The D327N mutation of human SHBG is associated with a number of good prognostic factors in breast cancer like estrogen receptor positivity and erb2 negativity. The identification of this mutation, that requires only a small sample of circulating blood, could be helpful whenever tissue samples are too scanty for the determination of prognostic factors, e.g. at fine needle aspiration for cytology. The search for this mutation is routinely performed in our laboratory with the Hinfl restriction fragment length polymorphism (RFLP) technique on polymerase chain reaction (PCR)-amplified DNA. The present report describes a new and simple enzyme-linked immunosorbent assay (ELISA) method to detect the SHBG mutation. The DNA enzyme immuno assay (DEIA) method, that is widely used in virology and has already been used to identify single nucleotide mutations, allows the identification of hybrids between specific DNA sequences and biotynilated probes with a monoclonal antibody against double stranded-DNA. We here report that by using two specific probes, specifically built for this kind of test, one complementary to the wild type SHBG sequence and the other to the D327N mutated DNA, the DEIA technique can be used to identify on PCR-amplified DNA the D327N mutation of human SHBG exactly as the Hinfl RFLP technique. The DEIA technique could, thus, be especially helpful when a large number of samples has to be processed, the technique being easier than Hinfl RFLP, specific, reproducible and allowing substantial time saving.     

Localization of chlorotetracycline fluorescence in human polymorphonuclear neutrophils

Chlorotetracycline (CTC) has been used in many cells as a probe for membranous calcium. In polymorphonuclear neutrophils (PMN), the changes in CTC fluorescence upon stimulation are considered to monitor an early event in the activation process. Using quantitative video-enhanced microscopy, we report that in resting cells about 80% of the CTC signal emanates from the perinuclear region of the cell, indicating that internal structures are labeled with CTC. Approximately 20% of the total CTC fluorescence is taken up in a compartment sensitive to mitochondrial inhibitors, which is not present in neutrophils depleted of nucleus and granules or cytoplasts. Upon stimulation PMN loaded with CTC exhibit a rapid, biphasic decrease in fluorescence that is dose dependent. The second phase of the response is not seen in neutrophil cytoplasts. These results suggest that internal stores of CTC are responsive upon stimulation and could account for the later decrease in CTC fluorescence, whereas the early phase of CTC changes represents the plasma membrane response.     

The nature of the vitamin B12 binding protein in human serum

It has been known for some years that the vitamin B₁₂ circulating in the plasma is bound to protein. When this plasma binding capacity is exceeded, by the oral or intravenous administration of large doses of the vitamin, the excess unbound vitamin is rapidly excreted in the urine (Mollin and Ross, 1953). The exact nature of this binding protein has yet to be identified. In normal subjects and in patients with myeloproliferative disorders it migrates electrophoretically with the speed of an α₁-globulin, and is present in that fraction of the serum proteins which is not precipitated by 0.6 M-perchloric acid, the seromucoid fraction (Weinstein, Weissman and Watkin, 1959). This serum fraction contains a number of antigenically distinct proteins (Burtin, 1964), two of which have been identified respectively as Orosomucoid and the α₁-acid–glycoprotein of Schultze (Hardwicke and St. Cyr, 1961). In this paper the binding of vitamin B₁₂ to the constituent fractions of a seromucoid, obtained by (NH₄)₂ SO₄ fractionation of serum, has been investigated by chromatography on DEAE-cellulose. Binding into this seromucoid fraction of the serum proteins is confirmed, in normals and in myeloproliferative disorders, but this binding appears to be onto two separable serum proteins which are neither the Orosomucoid nor any other recognized α₁-glycoprotein.     

Effect of insulin-like growth factor-type I (IGF-I) and insulin on the secretion of sex hormone binding globulin and IGF-I binding protein (IBP-I) by human hepatoma cells.

Dietary factors are known to modulate concentrations of sex hormone-binding globulin (SHBG). In the present study we have investigated the possibility that insulin like growth factor-type I (IGF-I) may be an additional regulator of SHBG using cultured human hepatoma cells which secrete SHBG. The inhibitory effect of insulin on SHBG secretion by these cells was confirmed but, in addition, IGF-I was shown to inhibit SHBG secretion by about 40% at a concentration of 100 nmol/l. A similar degree of inhibition was achieved using insulin at a concentration of 10 mumol/l. Insulin, but not IGF-I, was also found to inhibit the secretion of a low molecular weight IGF-binding protein (IBP-I), which is also secreted by hepatoma cells. It is concluded that IGF-I is an additional regulator of SHBG secretion by these cells and that it may be involved in regulating SHBG secretion in vivo in response to dietary factors.     

Serum vitamin D metabolites and their binding protein in patients with liver cirrhosis

Serum parameters of calcium metabolism were measured in 32 consecutive patients with biopsy-proven cirrhosis due to either hepatitis (n = 13), alcohol abuse (n = 11), Wilson's disease (n = 3), or primary or secondary biliary cirrhosis (n = 5). All measurements were normal in the small group of patients with Wilson's disease. The serum concentrations of albumin, vitamin D-binding protein, total calcium, phosphorus, and 1,25-dihydroxyvitamin D3 (1,25-(OH2)D3) were decreased in the other patients with cirrhosis, but their mean serum concentrations of ionized calcium, 25-hydroxyvitamin D3 (25-OHD3) and free 1,25-(OH2)D3 index were normal. A slight but significant increase in the serum PTH measured using a carboxyl-terminal antiserum was found. A significant correlation was found between the serum concentration of either albumin or vitamin D-binding protein and the serum concentrations of total calcium, 25-OHD3, 1,25-(OH2)D3, and PTH but not with ionized calcium or free 1,25-(OH2)D3 index. The observed abnormalities of calcium metabolism in unselected patients with cirrhosis were mainly due to decreased protein synthesis. Only the patients with severe cirrhosis had decreased concentrations of 25-OHD3 but they were nevertheless able to maintain a normal ionized serum calcium and free 1,25-(OH2)D3 level, possibly by means of compensatory hyperparathyroidism.     

2型糖尿病患者血清与尿维生素 D 结合蛋白水平的变化

目的：检测2型糖尿病患者血清与尿维生素 D 结合蛋白(VDBP)浓度的变化,探讨其临床意义。方法采用 ELISA 法检测102名健康对照组、106例2型糖尿病组患者血清 VDBP、尿 VDBP 与尿肌酐(Cr)比值及尿微量白蛋白与尿肌酐比值(UACR)。将2型糖尿病患者分为血糖控制良好组与血糖控制不佳组,正常白蛋白尿组与微量白蛋白尿组进行分析与比较。结果血清 VDBP 的正常参考上限为60.6μg/ ml,尿 VDBP/ Cr 的正常参考上限7.76 mg/ g。2型糖尿病组血清 VDBP 及尿 VDBP/ Cr 水平显著高于健康对照组[血：(44.83±6.65)μg/ ml 对(330.92±49.28)μg/ ml,尿：(5.55±2.89) mg/ g 对(15.19±6.38) mg/ g,均 P<0.01],其中血糖控制不佳组血清 VDBP 及尿 VDBP/ Cr 水平显著高于血糖控制良好组(P<0.05),微量白蛋白尿组尿 VDBP/ Cr 水平显著高于正常白蛋白尿组(P<0.01)。尿 VDBP/ Cr 对早期2型糖尿病肾病的诊断敏感性为96.4%,特异性为68%,准确性为83%。结论检测血清 VDBP 水平对于评估糖尿病病情有一定参考价值,联合检测尿 VDBP/ Cr 与 UACR 对于糖尿病肾病的早期诊断有重要的临床意义。     

Relationships among vitamin D, 25-hydroxyvitamin D, and vitamin D-binding protein concentrations in the plasma and milk of human subjects

We measured plasma and milk concentrations of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3), and vitamin D-binding protein (DBP) in a group of lactating women. All vitamin D compounds were quantitated using competitive protein binding assay, while DBP concentrations were determined by rocket electrophoresis. Vitamin D3 was the most abundant vitamin D compound in human milk, followed by vitamin D2, 25OHD3, and, finally, 25OHD2. The average vitamin D activity in milk was between 33-68 IU/liter, depending on the criterion of biological activity used. DBP concentrations in milk were approximately 3% of those in plasma. Significant relationships were found between plasma and milk levels for all vitamin D compounds. The milk to blood concentration ratio was greatest for vitamin D2, followed by vitamin D3, 25OHD2, and 25OHD3. (Thus, the parent compounds gained access into milk in a much more efficient fashion than their 25-hydroxy metabolites. It is postulated that this differential translocation is controlled by the DBP in the circulation.) There was no significant correlation between plasma and milk DBP concentrations, nor were milk DBP concentrations related to the vitamin D content of milk. This investigation supports the concept that the nutritional status of lactating mothers affects the vitamin D sterol potential of her milk which, in turn, would likely have an effect on the vitamin D status of her nursing infant.     

Ontogeny and effect of vitamin D deprivation on rat serum 25-hydroxyvitamin D binding protein.

Specific serum binding of 25-hydroxy-cholecalciferol (25-OHD3) was measured by saturation analysis in rats of various ages, and during vitamin D deprivation. The serum binding capacity for 25-OHD3 was observed to increase until age 6-8 weeks, then decline and remain stable thereafter at 3.7 x 10(-6) M. The 25-hydroxyvitamin D (25-OHD) concentration decreased in serum from rats fed vitamin D-free diet (t1/2 = 7 days). Rats fed 2 IU of vitamin D3/g of diet maintained stable serum levels of 25-OHD at 10-12 ng/ml. Serum binding capacity and affinity for 25-OHD3 was not affected by vitamin D deprivation or hypocalcemia. In addition, the binding affinity did not differ as a function of age (Kd = 3.3 x 10(-9) M). Since normal serum concentrations of 25-OHD in the rat are 2-5 x 10(-8) M, only 1-2% of the serum binding sites for this sterol are occupied under physiological conditions.