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Biomedical subjects

H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 253 records · Page 14Linked to original sources
PubMed1985

1H nuclear magnetic resonance studies of the conformations of vitamin D compounds in various solvents.

Using proton NMR, the solution conformation of the A ring of vitamin D3 and its analogs has been studied by application of the Karplus relation to the observed coupling constants. The A-ring conformations of vitamins D3, D2, and 25-hydroxyvitamin D3 were found to be solvent dependent, with a clear preference for an equatorial hydroxyl group in polar solvents such as methanol and dimethyl sulfoxide. Conversion of the hydroxyl group to an acetate did not affect solution conformation appreciably, but the corresponding t-butyl-dimethylsilyl ether derivative of vitamin D3 showed a strong preference for the 3 beta-equatorial conformer. The A-ring conformation of the active hormone, 1,25-dihydroxyvitamin D3, which has two hydroxyl groups competing for the equatorial position, was found not to be solvent-dependent.

Calcitriol↗
PubMed1985

The action of 1 alpha,25-dihydroxyvitamin D3 on rat small intestine: changes in translatable mRNA species.

The time course of changes in translatable mRNA from small intestinal mucosa was studied in vitamin D-deficient rats following an intrajugular dose of 1,25-dihydroxyvitamin D3. L-[35S]Methionine-labeled translation products were analyzed by high-resolution one- and two-dimensional electrophoresis. Changes were detected in two distinct proteins, Mr 14,000/pI 6.24 (Band I), and Mr 5,200/pI 4.86 (Band II). These increased to maxima of 1-2% each of the total in vitro-synthesized protein at 12 h after dosing. This represented a 3- and 10-fold increase, respectively, over control values. The time course of these changes suggests that regulation of the levels of the mRNAs coding for these proteins is not involved in the initial (6 h) peak of 1,25-dihydroxyvitamin D3-stimulated intestinal calcium transport in the rat.

Animals↗
PubMed1985

The effect of 1 alpha-hydroxyvitamin D2 on calcium metabolism in glucocorticoid-treated rats.

Intestinal calcium transport and renal calcium and phosphate excretion have been measured in normal and prednisolone-treated rats. Prednisolone treatment reduced intestinal calcium transport and increased phosphate excretion, causing a reduction in serum phosphate. Both 1 alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3 normalized phosphate excretion, whereas 1 alpha-hydroxyvitamin D3 was not fully effective in serum phosphate. Prednisolone treatment also caused a decreased bone density that was prevented both with 1 alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3. 1 alpha-hydroxyvitamin D2 appeared at least as effective as 1 alpha-hydroxyvitamin D3 in correcting the bone disturbance.

Animals↗
PubMed1985

Activation of retinoic acid by coenzyme A for the formation of ethyl retinoate.

All-trans-retinoic acid is metabolized to a less polar metabolite in rat liver microsomes. This metabolite was proven to be ethyl retinoate by cochromatography on high-performance liquid chromatography, base hydrolysis to all-trans-retinoic acid, and gas chromatography/mass spectral analysis. The formation of ethyl retinoate is a specific enzymatic process; the apparent Km for all-trans-retinoic acid is 9.8 microM. The production of ethyl retinoate is greatly stimulated by the addition of coenzyme A, suggesting the formation of a retinoic acid-coenzyme A intermediate (retinoyl-coenzyme A).

Adenosine Triphosphate↗
PubMed1985

Differences in the side-chain metabolism of vitamin D3 between chickens and rats.

In vitro metabolism of 25-hydroxy-24-oxovitamin D3 was studied in kidney homogenates from vitamin D-supplemented chickens and rats. In chicken homogenates, 25-hydroxy-24-oxovitamin D3 was converted predominantly to 23,25-dihydroxy-24-oxovitamin D3, 24,25-dihydroxyvitamin D3, and 23,24,25-trihydroxyvitamin D3. In rat homogenates, 25-hydroxy-24-oxovitamin D3 was not converted to either 24,25-dihydroxyvitamin D3 or 23,24,25-trihydroxyvitamin D3, but it was converted to 23,25-dihydroxy-24-oxovitamin D3 and 23-hydroxy-24,25,26,27-tetranorvitamin D3. The latter metabolite was not produced by the chicken preparations. The stereochemical configuration at C-24 of the 24,25-dihydroxyvitamin D3 produced by chicken homogenates was determined to be S. This contrasts with the R configuration of 24,25-dihydroxyvitamin D3 produced by 24-hydroxylation of 25-hydroxyvitamin D3. These results suggest that chickens have an enzyme that can reduce the 24-oxo group to 24S-hydroxyl group, whereas rats do not.

Animals↗
PubMed1985

Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting.

Identification of the porcine 1,25-dihydroxyvitamin D3 receptor protein on NaDodSO4/polyacrylamide slab gels was accomplished by two separate techniques: (i) assay of the specific binding activity of tritiated 1,25-dihydroxyvitamin D3 to protein eluted from NaDodSO4/polyacrylamide gels and renatured and (ii) immunoblotting of the partially purified receptor using two anti-receptor monoclonal antibodies. The porcine receptor preparation used in these studies was isolated from a crude nuclear extract of intestinal mucosa followed by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration HPLC, and DEAE-Sepharose chromatography. These receptor fractions were then electrophoresed on NaDodSO4/polyacrylamide gels. The receptor was eluted from the gel, renatured, and assayed for its ability to bind tritiated 1,25-dihydroxyvitamin D3. The renatured receptor appears as a single peak of specific tritiated 1,25-dihydroxyvitamin D3 binding activity. This binding activity corresponds to a band on a silver-stained gel that correlates with the receptor peak eluted from the DEAE-Sepharose column. It also corresponds to the highest molecular weight species identified on an immunoblot with anti-receptor monoclonal antibodies. The 1,25-dihydroxyvitamin D3 receptor protein has a molecular weight of 55,000 as deduced from its migration on NaDodSO4/polyacrylamide gels.

Animals↗
PubMed1985

Monoclonal antibodies to human vitamin D-binding protein.

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays.

Animals↗
PubMed1985

Regulation of bone mineral loss during lactation.

The effects of varying dietary calcium and phosphorus content, vitamin D deficiency, oophorectomy, adrenalectomy, and simultaneous pregnancy on bone mineral loss during lactation were examined in rats. Unless otherwise stated, the diet contained 0.47% calcium and 0.3% phosphorus and the rats were given 26 nmol of vitamin D3. Femur ash weights were determined after 21 days of lactation and on age-matched nonlactating rats. Decreasing dietary calcium to 0.02% caused an increased loss of bone mineral, whereas increasing dietary calcium to 1.4% increased plasma calcium levels to 12 mg/100 ml but did not diminish the bone mineral loss observed during lactation. Varying dietary phosphorus did not have a major effect on bone mineral loss during lactation. In vitamin D-deficient rats, bone mineral loss during lactation was independent of dietary calcium levels and slightly greater than the loss observed in vitamin D-replete rats fed the normal calcium diet. Oophorectomy and adrenalectomy did not produce changes in femur ash weights of nonlactating rats or reduce bone mineral loss during lactation. Rats mated during their postpartum estrus and thus simultaneously pregnant and lactating, lost the same amount of bone mineral as caused by lactation alone.

Adrenalectomy↗
PubMed1985

Effect of vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryos.

The biochemical nature of the physiological defect found in chick embryos from hens supported on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D is described. Vitamin D-deficient hens (44-wk-old) were divided into six groups of five and dosed daily for 19 wk with either 2.0 micrograms of 25-hydroxyvitamin D3, 2.0 micrograms of 24,24-difluoro-25-hydroxy-vitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3, 2.0 micrograms of 24,25-dihydroxyvitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3 plus 2.0 micrograms of 24,25-dihydroxyvitamin D3, or vehicle only. Normal embryonic development was found in eggs from hens given 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, whereas embryos from hens given 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or their combination were abnormal and failed to hatch. Embryos from hens fed 1,25-dihydroxyvitamin D3 and/or 24,25-dihydroxyvitamin D3 had vitamin D deficiency: low bone ash, low plasma calcium, low total body calcium, and extremely high plasma phosphorus. Because the shell is the major source of calcium for the developing embryo, calcium transport from the shell to the embryos across the chorioallantoic membrane apparently fails, giving rise to the observed defects in embryonic development.

24,25-Dihydroxyvitamin D 3↗
PubMed1985

1 alpha-Hydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat.

An LD50 of 0.2 mg/kg body wt has been determined for 1 alpha-hydroxyvitamin D3 in the rat. In comparison, the LD50 for 1 alpha-hydroxyvitamin D2 is between 3.5 and 6.5 mg/kg. In terms of chronic toxicity, 1 alpha-hydroxyvitamin D3 at a dose of 5 micrograms/kg/day causes death of one-half the animals in a 4-week period. On the other hand, 20 micrograms/kg/day of 1 alpha-hydroxyvitamin D2 is required to induce similar toxicity. The body weight record and renal calcium accumulation during chronic treatment support the above conclusion. It therefore appears that 1 alpha-hydroxyvitamin D2 is between 5 and 15 times less toxic than 1 alpha-hydroxyvitamin D3. This surprising result prompted a reexamination of the relative biological activity of 1 alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3. Both compounds are equally potent in the stimulation of intestinal calcium transport, bone calcium mobilization, in the elevation of serum phosphorus, and in the healing of rickets in the rat. The reason for lower toxicity of 1 alpha-hydroxyvitamin D2 is unknown. The results suggest that 1 alpha-hydroxyvitamin D2 might represent a therapeutically superior compound.

Animals↗
PubMed1985

Vitamin D-dependent calcium transport.

Vitamin D must be metabolized to 25-OH-D3 or calcidiol in the liver and subsequently to 1,25-(OH)2D3 (calcitriol) in the kidney to produce its physiological actions. Calcitriol stimulates calcium and phosphorus transport reactions in the small intestine and together with parathyroid hormone stimulates calcium transport reactions in bone and kidney. In the small intestine calcitriol brings about its response in a complex manner. It provides a very rapid response on existing villus cells, causing a rise in calcium transport within 6 h postadministration. This transport response subsides by 18 h, and a second response makes its appearance at 24-48 h. This second response is likely to be an action on crypt cells which then migrate into the villus region and bring about calcium transport. The molecular mechanism of calcium transport in the villus cells has been examined. Calcitriol localizes specifically in the nucleus within 0.5 h postadministration, and it does not localize in any of the other cell fractions. A receptor has been discovered for calcitriol, and interaction with this receptor is required for calcitriol to bring about its action in stimulating calcium transport. It is therefore believed that calcitriol, together with receptor, binds to specific portions of nuclear DNA to bring about transcription of specific genes that code for calcium and phosphorus transport proteins. Only one protein induced by calcitriol has been described. It is the calcium-binding protein found in the cytoplasm. Exactly how it functions in calcium transport is in debate, although the time sequence of its appearance appears to be consistent with a role in that system. It is likely that other gene products are induced by calcitriol, and they are currently being characterized. In the neonatal rat, active calcium transport does not make its appearance until 14-16 days postpartum. The intestine is not sensitive to vitamin D or calcitriol prior to this time. This lack of sensitivity is because of the lack of a receptor for calcitriol. The receptor to calcitriol can be precociously induced by hydrocortisone injections, or it can be delayed by adrenalectomy. Incubation of intestine from neonatal rat pups at 14 days postpartum with hydrocortisone brings about in vitro appearance of the receptor molecule. These results and results of studies involving genetic vitamin D resistance strongly suggest that calcitriol must function by interaction with the receptor, presumably by a nuclear mechanism, to bring about intestinal calcium transport. The molecular mechanism, however, remains to be described.

Animals↗
PubMed1984

Multiple forms of vitamin D-dependent calcium-binding protein in rat kidney.

The technique of two-dimensional electrophoresis was used in combination with a highly sensitive silver stain to study vitamin D-dependent calcium-binding protein (CaBP) in rat kidney. Rat renal CaBP was shown to co-migrate almost exactly with CaBP purified from chick intestine suggesting evolutionary conservation of this protein. In some cases rat renal CaBP appeared not as a single polypeptide, but rather as a cluster of 4 polypeptides. Formation of the satellite cluster of CaBP in response to high doses of 1,25-dihydroxyvitamin D3 occurred in young rats which had been maintained on a vitamin D-deficient diet for 2 weeks, as well as in older rats which had been maintained on the same diet for 5 months. The 4 forms of CaBP were not the result of various states of Ca2+ binding, but rather the result of an enzymatic reaction. This was shown by 3 experiments. 1) Adding excess EGTA to samples containing the 4 satellite forms did not change the two-dimensional electrophoretogram. 2) Incubation of purified chick intestinal CaBP with kidney cytosols from D-deficient rats brought about the formation of the satellite CaBP forms from the chick protein. However, purified chick CaBP was unchanged by incubation in buffer alone for up to 2 h at 37 degrees C. 3) Placing rat kidney cytosols in a boiling water bath for 10 min inactivated the factor which generated the satellite forms as would be expected for an enzyme. The physiological significance of these forms of CaBP is as yet unknown.

Animals↗
PubMed1984

Chemical synthesis of (24R)-24,25-dihydroxy[26,27-3H]vitamin D3 of high specific activity.

Chemical synthesis of (24R)-24,25-dihydroxy-[26,27-3H]vitamin D3, and its 24-epimer has been devised that allows introduction of 3H at the terminal step of the synthesis. The epimeric mixture is derivatized as the tris(trimethylsilyl) ethers and resolved by high-performance liquid chromatography. The product has a specific activity of 178 Ci/mmol and is fully active in binding to the rat plasma vitamin D binding protein and in the elevation of serum calcium levels of vitamin D deficient rats. The synthesis begins with the readily available 3 beta-hydroxy-5-cholenic acid methyl ester and involves a Pummerer rearrangement, introduction of the delta 7, irradiation, and isolation of the 26,27-dinor-25-carboxylic acid methyl ester of vitamin D3. This compound is then treated with a Grignard reagent containing 3H (80 +/- 10 Ci/mmol).

24,25-Dihydroxyvitamin D 3↗
PubMed1984

The early time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 as determined by computer analysis of two-dimensional electrophoresis gels.

The time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 was examined in embryonic chick duodena by single-label autoradiography. Duodena were excised from 19-day-old embryos, cultured for 24 h in defined medium, and exposed to 1,25-dihydroxyvitamin D3 at 1/2, 1, 1 1/2, 2, 2 1/2, 4, 6, and 20 h before the end of the culture period. Control duodena were identically handled but were not exposed to the hormone. All duodena were pulsed with [14C]leucine during the final 30 min of incubation. Cytosolic proteins, extracted from each radiolabeled tissue, were resolved by two-dimensional electrophoresis and autoradiographs were prepared from the resulting gels. Calcium-binding protein was detectable by autoradiography in duodena cultured with hormone for 1 h or more but not in the control duodena. The autoradiographs were converted to digital images by a scanning densitometer and entered into the Man-computer Interactive Data Access System. The total radioactivity incorporated into calcium-binding protein during the 30-min pulse was determined by computer analysis of the digitized autoradiographs, and the rate of calcium-binding protein biosynthesis was calculated. Duodena cultured with hormone for 1-20 h synthesized calcium-binding protein at rates of 129 +/- 17 to 8800 +/- 110 pg/mg of cytosolic protein/30 min. These rates demonstrate that calcium-binding protein is induced within the first hour of exposure to 1,25-dihydroxyvitamin D3, but in amounts too small to be detected by commonly used immunoassays.

Animals↗
PubMed1984

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3.

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.

Animals↗
PubMed1984

Topographical and developmental studies on target sites of 1,25 (OH)2 vitamin D3 in skin.

Tritium-labeled 1,25 (OH)2 vitamin D3, when injected into vitamin D-deficient adult and pregnant rats is concentrated and retained strongest in nuclei of cells in the outer root sheath of the hair, followed by the stratum granulosum, spinosum, and basale of the epidermis. In the hair follicle, in addition to the most heavily labeled outer root sheath, nuclear labeling exists also in cells of the hair bulb and of the inner root sheath, as well as in basal cells of the sebaceous gland. In contrast, cells of the dermal papilla and the connective tissue of the dermis are generally unlabeled, except for labeled cells in the outer connective tissue sheath at the infundibulum of vibrissae of 20-day fetal rats and a few scattered labeled cells in the dermis, probably macrophages. In the developing hair, in 18- and 20-day fetal rats, a distinct topographic pattern of labeled cells can be seen, which is characteristic of the different stages of hair follicle development. In the hair germ, heavily labeled cells appear first in the stratum spinosum. In the hair peg, they remain in this position in its juxtaepidermal portion; however, when a dermal papilla develops, heavily labeled cells assume a marginal position. This suggests a sequential epidermal-epidermal and mesenchymal-epidermal receptor induction. Injection of tritium labeled 25 (OH) vitamin D3 did not show nuclear concentration in these tissues and excess unlabeled 25 (OH) vitamin D3--unlike excess 1,25 (OH)2 vitamin D3--did not prevent nuclear uptake of tritium labeled 1,25 (OH)2 vitamin D3. The results indicate differential effects of 1,25 (OH)2 vitamin D3 on different structures in the epidermis and dermis.

Aging↗