Radioimmunoassay of the binding protein for vitamin D and its metabolites in human serum: concentrations in normal subjects and patients with disorders of mineral homeostasis.

A radioimmunoassay for the binding protein for vitamin D and its metabolites (DBP) has been developed. Suitable rabbit anti-DBP antiserum was elicited after primary and one booster injection. Anti-DBP antisera, as well as antigroup-specific component antisera, produced a single, monospecific line of percipitation when reacted against purified DBP and human serum. DBP was iodinated with 125I and 125I-DBP was purified by gel filtration on Sephadex G-200. Binding of 125I-DBP by 20 nl of rabbit anti-DBP antisera was approximately 50% and was sharply competed for by 0.4-4.0 ng of DBP standard. Displacement of 125I-DBP by human serum dilutions or standard DBP gave identical curves, and only weak competition was observed with old and new world primate sera. Apo- and holo-DBP possessed indistinguishable immunoreactivity. The assay detects DBP in 1-10 nl of human serum with reasonable accuracy and with reasonable intra- and interassay precision. The mean serum concentration (+/- SEM) for a group of 40 normal adults was 525 +/- 24 mug/ml and no sex difference was observed. Higher levels were found in sera from pregnant women and women receiving oral contraceptives, and decreased concentrations were observed in premature cord and hypoproteinemic sera. No significant correlation between serum DBP levels and serum 25-hydroxycalciferol levels was found, and the DBP content of sera from vitamin D-deprived and vitamin D-treated subjects was indistinguishable from that of normal adults. DBP accounts for 6- of the alpha globulin in normal human serum. Considering the normal serum content of the parent vitamin and its metabolites to be approximately 0.1-0.2 mum, these immunoassay data confirm previous saturation analyses of human serum antiricketic sterol-binding capacity and suggest that greater than 95% of DBP circulates as the apoprotein under normal conditions.

Vitamin D metabolite-binding proteins in human tissue.

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD3) and 1, 25-dihydroxycholecalciferol (1,25-(OH)2D3). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD3-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport alpha-globulin. A cathodal, 1, 25-(OH)2D3-binding protein, which sedimented at 3-4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.

Competition by 24,25-dihydroxycholecalciferol in the competitive protein binding radioassay of 25-hydroxycalciferol.

24,25-dihydroxycholecalciferol (24,25-(OH)2D3) is equipotent to 25-hydroxycholecalciferol in the displacement of 3H25-OHD3 from rat serum binding sites, and is extracted by the ethanol procedure recommended in non-chromatographic competitive protein binding radioassays for 25-OHD. Serum 24,25-(OH)2D content was measured following 24,25-(OH)2D3 isolation from lipid extracts by liquid-gel partition chromatography. Since normal serum 24,25-(OH)2D concentration is appreciable, non-chromatographic 25-OHD assays most probably overestimate serum 25-OHD levels as a result of their recognition of 24,25-(OH)2D3.

Local distinguishability of multipartite orthogonal quantum states

We consider one copy of a quantum system prepared in one of two orthogonal pure states, entangled or otherwise, and distributed between any number of parties. We demonstrate that it is possible to identify which of these two states the system is in by means of local operations and classical communication alone. The protocol we outline is both completely reliable and completely general; it will correctly distinguish any two orthogonal states 100% of the time.

25-Hydroxyvitamin D transport in human plasma. Isolation and partial characterization of calcifidiol-binding protein.

The binding protein for 25-hydroxycholecalciferol (25-OH-D3 or calcifidiol) in human plasma has been purified from Cohn Fraction IV. Following in vitro labeling with 25-OH-[3H]D3, the isolation sequence of procedures included: DEAE-cellulose chromatography; gel filtration on Sephadex G-200; chromatography on DEAE-Sephadex; preparative polyacrylamide gel electrophoresis. These procedures resulted in a calcifidiol-binding protein (Cal-BP) which had been purified approximately 170-fold, and which was homogeneous by physical and immunological criteria. The purified Cal-BP had inter-alpha mobility, a sedimentation constant (S20, w) of 3.46 S, and a molecular weight of approximately 59,000. Sucrose gradient ultracentrifugation of Cal-BP and ligand incubations indicated that there was apparently one binding site for either cholecalciferol, calcifidiol or 1,25-dihydroxycholecalciferol per molecule of human Cal-BP. The protein had highest affinity for calcifidiol, displaying an apparent dissociation constant (Kd) of 6.4 X 10(-8) M for this sterol. Specific anti-human Cal-BP antisera were prepared in rabbits, and produced precipitate lines of identity between Cal-BP and human serum. Specific binding of vitamin D3,25-OH-D3, and 1,25-(OH)2D3 by human serum was completely neutralized after immunoprecipitation of the serum with the gamma globulin fraction of anti-Cal-BP antiserum, indicating a common transport protein for these sterols in human plasma. There was no immunological cross-reactivity between Cal-BP and rat or chicken sera, indicating that the Cal-BP in these three sera are immunologically completely distinct. Purified human Cal-BP and human sera also produced lines of identity with commercial anti-human group-specific component (Gc) antisera in radial immunodiffusion experiments. This finding supports an earlier report of the identity of calciferol/calcifidiol-binding protein and group-specific component in human serum. The Cal-BP content of human serum is approximately 10(-5) M, whereas the calcifidiol content is approximately 10(-7) M. Normally, the dominant moiety of human plasma Cal-BP is the apoprotein.

Affinity chromatography with 25-hydroxycholecalciferol ester in the isolation of the binding protein for vitamin D and its metabolites from human serum.

The 3 beta-hemisuccinate ester of 25-hydroxycholecalciferol was prepared by incubating the sterol with succinic acid anhydride in the presence of 4-dimethylaminopyridine at 4 degrees. The ester was isolated by silica gel column chromatography, and its hydrolysis in ethanolic sodium hydroxide yielded 25-hydroxycholecalciferol. The ester was covalently linked to aminoagarose in the presence of isobutylchloroformate, yielding 16 to 60 micrograms of sterol per packed ml of gel. Normal human serum was applied to the 3 beta-hemisuccinate-25-OH-D3 aminoagarose gel and elutions with buffer of increasing molarity were carried out. Total protein and DBP content of the 6M guanidine elution fractions revealed a 12 to 134-fold purification of human serum DBP by this technique. Best purifications were achieved by incubating serum with the gel at pH 8.3 and rinsing the gel with 0.3 M KCl prior to its elution with the guanidine. Our results indicate that affinity chromatography with this ligand, or possibly other vitamin D sterol esters, is a practical and useful technique in the analysis and preparative isolation of vitamin D sterol-binding proteins.

Effect of calcium therapy in the sick premature infant with early neonatal hypocalcemia.

Twenty-seven sick premature infants with serum calcium concentrations less than 6.0 mg/dl during the first day of age were enrolled in a prospective controlled study involving two treatment regimens--calcium given as a bolus or a drip--or no treatment. Mean total calcium concentration was 5.5 +/- 0.8 mg/dl, and ionized calcium was 3.1 +/- .3 mg/dl, with no significant difference between treatment groups. By 24 hours, in all groups total calcium had increased to greater than 6.0 mg/dl (bolus 6.5 +/- 1.1, drip 7.0 +/- 0.4, control 6.6 +/- 0.4) and ionized calcium to greater than 3.5 mg/dl (bolus 3.9 +/- 0.3, drip 3.6 +/- 0.6, control 3.6 +/- 0.3). Ionized and total calcium concentrations were significantly correlated (r = 0.562; P less than 0.001), but total calcium did not predict ionized calcium in any group. These data support the concept that, even in sick infants, early neonatal hypocalcemia is a physiologic phenomenon that may not require treatment.

Serum calcitonin concentrations in premature infants during the first 12 weeks of life.

Twenty-eight premature infants of mean gestation 30.9 +/- 2.5 weeks and mean birth weight 1175 +/- 206 g had repeated serum calcitonin concentrations determined over the first 12 weeks of life. Serum calcitonin concentrations slowly fell but remained elevated even at 12 weeks of age [normal adult = 71 +/- 48, 1 week (N = 15) = 327 +/- 167, 3 week (N = 23) = 270 +/- 129, 6 week (N = 16) = 249 +/- 154, 9 week (N = 13) = 214 +/- 108, 12 week (N = 12) = 174 +/- 11]. Throughout this period, serum total calcium was normal or low (8.4 +/- .8-9.3 +/- 1.0). Serum phosphorus was normal or low (6.0 +/- 1.4-6.5 +/- 1.0), and serum magnesium was normal (1.7 +/- 0.24-1.8 +/- 0.34). The reason for the sustained elevation of serum calcitonin in these very small, sick, premature infants in unclear.