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At least 343 records · Page 19Linked to original sources
PubMed1989

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein.

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.

Animals↗
PubMed1990

Separation of the cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3.

The cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 have been separated. The cytochrome P-450 fractions required NADPH, mitochondrial ferredoxin and ferredoxin reductase for catalytic activity. The present report demonstrates that different forms of cytochrome P-450 are involved in 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 and provides a basis for further purification and characterization of these enzymes.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1991

Effect of active vitamin D3 on the levels of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine-binding protein.

Effect of 1 alpha-OH-vitamin D3 (1 alpha-OH-D3) and 1,25-(OH)2-vitamin D3 (1,25-(OH)2-dihydroxycholecalciferol)(1,25-(OH)2-D3) on the levels of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) was studied in rats and cultured dRLh cells. Deprivation of rats from vitamin D decreased the activity of cytosolic NADPH-dependent T3 binding in rat kidney and liver. The decrease was restored by administration of 1 alpha-OH-D3(0.2 micrograms/kg). The activity of cytosolic NADPH-dependent T3 binding was increased in the dRLh cells by addition of 1,25-(OH)2-D3 to the culture medium. The maximal binding capacity (MBC) was increased by 1,25-(OH)2-D3 without changes in the affinity constant. These results suggested that active vitamin D3 plays an important role in the regulation of cellular T3 translocation through increasing the binding capacity of NADPH-dependent cytosolic T3-binding protein.

Animals↗
PubMed1984

Circannual versus seasonal variations of longitudinally sampled 25-hydroxycholecalciferol serum levels.

Seasonal variations in human serum levels of 25-hydroxycholecalciferol (25OHD) have been largely documented in transverse studies of population. But seasonality is not per se a demonstration that 25OHD serum levels fluctuate along the course of year according to a waveform profile with a periodic rhythm. Because of this, we attempted to investigate the possible occurrence of a circannual rhythm for 25OHD serum levels in a longitudinal design, by fitting a 365.25-day cosine curve to temporal biodata recorded in 10 clinically healthy subjects, monthly sampled for RIA determinations of 25OHD. Cosinor procedure statistically validated the occurrence of a circannual rhythm for 25OHD serum concentrations at a highly significant level of probability (P = 0.0015) for null hypothesis amplitude = 0. With 95% of probability, amplitude ranges from 5.0 to 16.5 ng/ml (mean value of oscillation = 10 ng/ml), while acrophase is temporally located from September 14 to December 3 (mean timing = October 21). Yearly, mean values for 25OHD serum concentrations is of 40.3 +/- 5.4 ng/ml as quantified by the line which transversely divides the cosine curve interpolating original biodata. By calculating the band of a complete 12 months variability which includes 90% of the distribution with 90% confidence limits, the circannual chronodesm of 25OHD serum levels has been obtained. Such a chronodesmic sinusoid has been compared to the circannual chronogram. By this comparison, a dissociation between the crest (October) and the peak (August) has been detected. The finding suggests that seasonal variations are superimposed to the circannual rhythm. Seasonal but also circannual changes, thus, characterize the yearly variability of 25OHD serum levels in man.

Adult↗
PubMed1986

Hydroxycholecalciferols modulate parathyroid hormone and calcitonin sensitive adenylyl cyclase in bone and kidney of rats. A possible physiological role for 24,25-dihydroxy vitamin D3.

In particulate fractions from rat bone cells, but not from kidney, 24,25-(OH)2 D3 inhibits in a dose dependent manner (1 nM and above) the parathyroid hormone (PTH)-activated adenylyl cyclase. In contrast, 24,25-(OH)2D3 enhances the calcitonin (CT) stimulated cyclase in bone, but attenuates the CT-induced cyclase response in kidney. In supranormal concentrations 1,25-(OH)2D3 is also able to reduce the PTH-stimulated adenylyl cyclase in bone. In comparison, neither vitamin D3 metabolite interferes with stimulation of adenylyl cyclase from pituitary cell membranes by thyroliberin (TRH) or vasoactive intestinal polypeptide (VIP). These findings may have important therapeutical consequences in preventing excessive PTH action and bone demineralization.

24,25-Dihydroxyvitamin D 3↗
PubMed1980

Purification of plasma vitamin D metabolites for radioimmunoassay.

The ability of four solvent systems to extract tritiated 25-hydroxy vitamin D3, 24,25-dihydroxy vitamin D3, 25,26-dihydroxy vitamin D3 and 1 alpha, 25-dihydroxy vitamin D3 from plasma was compared. Diethyl ether gave the highest yield of metabolites and lowest yield of "lipid" materials, the dihydroxylated metabolites were more readily extracted than 25-hydroxy vitamin D3. Following extraction with ether the vitamin D metabolites could be separated, without prior purification, on a 6.2 mm X 250 mm Zorbax-Sil high pressure liquid chromatography (HPLC) column, a simplification of previous methods. Plasma 1 alpha, 25-dihydroxy vitamin D levels measured after purification by this method were not significantly different from those obtained by an established, more complex method.

24,25-Dihydroxyvitamin D 3↗
PubMed1980

Quantitation of the main metabolites of vitamin D in a single serum sample. I. Extraction, separation and purification of metabolites.

A method for extraction, separation and purification of the main serum metabolites of vitamin D from a single serum sample is described. The method involved extraction of serum by diethylether and separation and purification of vitamin D, 25-OHD and the dihydroxymetabolites 24,25-(OH)2D, 25,26-(OH),2D and 1,25-(OH)2D by elution in three steps from a short open silicic acid column. The eluted vitamin D metabolites were further separated and purified by high pressure liquid chromatography (HPLC). The HPLC systems described separated the D2 and D3 forms of vitamin D, 25-OHD, 1,25-(OH)2D, and probably also 24,25-(OH)2D and 25,26-(OH)2D. The metabolites were purified by the methods described for further quantitation by UV-absorption or competitive protein binding assays, and were found to be homogenous on re-chromatography with different HPLC systems. Good recoveries were obtained for all the metabolites.

24,25-Dihydroxyvitamin D 3↗
PubMed1980

The estimation of vitamin D and some metabolites in human plasma by mass fragmentography.

A mass fragmentographic assay for vitamin D3, 25-hydroxyvitamin D3, 25-hydroxyvitamin D2 and 24,25-dihydroxyvitamin D3 is described. After extraction of plasma with methanol and dichloromethane, Lipidex 5000 chromatography was used to separate the plasma extract into three fractions (a vitamin D fraction, a 25-hydroxyvitamin D fraction and a 24,25-dihydroxyvitamin D fraction). Cholesterol was removed from the vitamin D fractio by thin-layer chromatography. :After addition of vitamin D2 to each fraction, isotachysterol trimethylsilyl ethers were formed. Recoveries prior to gas chromatography-mass spectrometry were assessed by the use of tritiated standards added to the plasma before extraction. Using this procedure, normal ranges were established (vitamin D3: 3--17.7, 25-hydroxyvitamin D3: 5.9--35.2, 25-hydroxyvitamin D2: 0.6---1.0 and 24,25-dihydroxyvitamin D3: 0.6--2.9 micrograms/l).

24,25-Dihydroxyvitamin D 3↗
PubMed1981

An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults.

A method for routine determination of vitamin D and its major metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxy-vitamin D (1,25(OH)2D) in serum samples from normal children and adults has been developed. Methodological improvements enable a rapid and accurate analysis of 25(OH)D and also the microscale screening of other metabolites present in large concentrations in serum. Vitamin D and its metabolites are extracted from serum samples using hexane/propan-2-ol, which allows a convenient separation of the water soluble and lipid soluble fractions from each other and also from proteins. Preparative silicic acid chromatography was used to separate vitamin D from its metabolites and also from the major portion of co-eluting lipid contaminants. An automated LC solvent delivery and sample introduction system was used to achieve the rapid separation of metabolites. Vitamin D was further purified using adsorption high-performance liquid chromatography and assayed using reverse phase high-performance liquid chromatography connected with UV detection. The 25(OH)D fraction from the preparative chromatography was measured by a competitive protein-binding assay along with 24,25(OH)2D, which was purified by reverse phase high-performance liquid chromatography along with 1,25(OH)2D. A diluted human serum from a pregnant woman (3rd trimester of pregnancy) was used as source of the binding protein for 25(OH)D and 24,25(OH)2D. 1,25(OH)2D was determined by a competitive protein-binding assay using a diluted cytoplasmic 1,25(OH)2D receptor protein isolated from the intestinal mucosa of rachitic chicks. Vitamin D and its metabolite levels were assayed in serum samples from normal children and adults.

24,25-Dihydroxyvitamin D 3↗
PubMed1982

A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid chromatographic step.

A simple one step high-performance liquid chromatographic (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D3 (25-OHD3) and 25-hydroxyvitamin D2 (25-OHD2) is described. Plasma (2-4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C18 cartridge, washed with methanol:water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD3, added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD3 was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD3) and between-(8.0% for 25-OHD3) batch reproducibilities were satisfactory.

25-Hydroxyvitamin D 2↗
PubMed1982

A simple and sensitive assay for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human serum.

An improved method is described which permits the simultaneous determination of 25-hydroxyvitamin D [25-(OH)D], 24,25-dihydroxyvitamin D [24,25-(OH)2D] and 1,25-dihydroxyvitamin D [1,25-(OH)2D] in milliliters of human serum. Methodological improvements enabled a rapid and almost complete extraction of the three metabolites from serum and omission of adding labeled internal standards to each serum sample for the calculation of individual recoveries. Commercially available stable chick embryo intestinal mucosa cytosol preparation made the troublesome preparation of cytosol receptor for 1,25-(OH)2D unnecessary. The procedure involves saturation of serum with ammonium carbonate and extraction with methanol/ethyl acetate, followed by separation of 25-(OH) D from the dihydroxy metabolites of vitamin D by Sephadex LH-20 column chromatography and further separation of the dihydroxy metabolites into 24,25-(OH)2D and 1,25-(OH)2D by high-pressure liquid chromatography. This is followed by individual determination of each metabolite by competitive protein-binding assay or radioreceptor assay.

24,25-Dihydroxyvitamin D 3↗
PubMed1983

Studies on the presence of 25-hydroxyvitamin D in human saliva.

Saliva collected from volunteers of both sexes at various times throughout the day was examined for the presence of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The method used was a specific high-performance liquid chromatographic procedure with a minimum detectable limit for 25-hydroxyvitamin D of 0.05 microgram/1, (130 pmol/1 for 25-hydroxyvitamin D3) using 20 ml of saliva. No 25-hydroxyvitamin D could be detected in any of the samples. Examination of larger quantities of saliva (100 ml) by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry showed the presence of a peak possibly derived from 25-hydroxyvitamin D3, at a concentration not in excess of 65 pmol/l. Using known binding characteristics of 25-hydroxyvitamin D and plasma vitamin D binding protein, and comparing them with similar characteristics for cortisol and plasma cortisol binding globulin and the known salivary concentration of cortisol, simple calculations indicated that a likely concentration of 25-hydroxyvitamin D in saliva would be around 8 pmol/l. Previously reported values ranging from 130-4160 pmol/l (50-1600 micrograms/l) using a simple competitive protein binding assay could not be confirmed.

25-Hydroxyvitamin D 2↗
PubMed1985

Determination of vitamin D binding protein by Scatchard analysis and estimation of a free 25-hydroxy-vitamin D index.

A simple method for the quantification of the vitamin D binding capacity (concentration of vitamin D binding protein, DBP) in serum is described. 25-Hydroxy-vitamin D can conveniently be measured by a binding protein assay utilizing dilute human serum as a source of DBP. The same methodology was used to determine the concentration of DBP by Scatchard analysis. In comparison to radial immunodiffusion, a correlation coefficient of 0.863 (p less than 0.001) was found with a regression line y = 0.963x + 0.08 (n = 52). Normal values were between 3 and 8 mumol/l, the coefficient of variation was less than 7% (median 4.9%). A simple routine version of this test requiring only 5 tubes per sample at a sample dilution of 1:15 000 had an interassay variation of less than 8%. The apparent affinity constants of DBP obtained by the Scatchard analysis had a very poor interassay reproducibility. Thus, the molar ratio of 25-hydroxy-vitamin D and DBP was used as a 'free 25-hydroxy-vitamin D index'. The normal range for a control group was 0.005-0.015, with samples taken in winter.

Binding, Competitive↗
PubMed1985

Rapid method for measuring physiological concentrations of 25-hydroxyvitamin D levels in blood serum.

A new single-step Extrelut column procedure for obtaining the 25-hydroxyvitamin D extract, suitable for a direct radiocompetitive test, is described. Separation of 0.1 ml of serum, on a small Extrelut column and radioassay allows a rapid, sensitive and accurate determination of 25-hydroxyvitamin D concentration. The detection limit of the determination was 2 nmol/l (0.8 microgram/l) of serum. The recovery of 250HD3 added to serum was 99-108%. Intra-assay and interassay CV values were 10 and 14%, respectively. Although the method does not permit separation of dihydroxylated metabolites of vitamin D from 25-hydroxyvitamin D, it is useful in clinical practice.

Binding, Competitive↗
PubMed1986

Purification of vitamin D binding protein from human plasma using high performance liquid chromatography.

Vitamin D binding protein/group-specific component was purified from human plasma by chromatographic techniques utilising high performance liquid chromatography and by traditional low pressure chromatographic techniques alone. Use of high performance liquid chromatography considerably reduced the time taken to prepare pure vitamin D binding protein and increased the yield to 16% compared with 2.8% using the traditional methods. The vitamin D binding protein prepared by high performance liquid chromatography was shown to be highly pure by amino acid sequence, SDS gel electrophoresis and by antibody production. The amino acid sequence was confirmed and extended. The affinity constants of the high pressure liquid chromatography purified vitamin D binding protein for 25 hydroxycholecalciferol (25 OHD3) and 1,25 dihydroxycholecalciferol 1,25(OH)2D3 were 1.9 X 10(7) mol/l and 2.6 X 10(6) mol/l, respectively.

Amino Acid Sequence↗
PubMed1986

Estimation of vitamin D3 and 25-hydroxyvitamin D3 in muscle and adipose tissue of rats and man.

An assay is described based on a high pressure liquid chromatography system for measurement of vitamin D3 and 25-hydroxyvitamin D3 concentration in adipose tissue and muscle. The sensitivity of the assay is less than 1 ng/g of tissue. Neither vitamin D3 nor 25-hydroxyvitamin D3 were found in tissues of vitamin D-deficient rats using this method. 25-Hydroxyvitamin D3 could not be detected in adipose tissue and very little was found in muscle of normal rats. The mean vitamin D3 concentrations in 15 samples of human perirenal adipose tissue was 45.3 ng/g +/- 22.2 (SD) and in 6 samples of axillary tissue 115.6 ng/g +/- 52.4 (SD). Human adipose tissue contains a substantial amount of vitamin D3 and its contribution to the maintenance of vitamin D status is discussed.

Adipose Tissue↗
PubMed1987

Vitamin D-binding protein synthesized by a human hepatocellular carcinoma cell line.

The binding protein for 25-hydroxycholecalciferol was studied in the medium spent for the culture of HuH-7 cells, which were originally derived from a human hepatocellular carcinoma tissue. The binding protein for 25-hydroxycholecalciferol synthesized by HuH-7 cells was immunologically similar to vitamin D-binding protein in human serum and had an inter-alpha mobility. A sedimentation coefficient of 4.1 S was found on sucrose density gradient analyses. The molecular weight was estimated to be approximately 58,000 by gel filtration on a standardized column of Sephadex G-150. When mixed with filamentous actin purified from rabbit skeletal muscle, it depolymerized filamentous actin and bound to monomeric, globular actin to make a 5.5 S 1:1 molar complex with a molecular weight of approximately 100,000. These results support the conclusion that HuH-7 cells produce a functional vitamin D-binding protein.

Calcifediol↗