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Biomedical subjects

M F Holick

Publications and source records attributed to M F Holick.

At least 127 records · Page 7Linked to original sources
PubMed1991

Synthesis of 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D binding protein.

Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.

Affinity Labels↗
PubMed1991

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.

Calcifediol↗
PubMed1990

Gas chromatography-mass spectrometry of conjugated dienes by derivatization with 4-methyl-1,2,4-triazoline-3,5-dione.

The dienophile 4-methyl-1,2,4-triazoline-3,5-dione forms stable adducts with conjugated dienes by generating Diels-Alder cycloaddition products. The reaction is rapid, highly selective for conjugated dienes and the derivatives are suitable for analysis by gas chromatography. Their mass spectra are marked by their simplicity and by the presence of abundant fragment ions diagnostic of the diene position in the parent compound.

Alkenes↗
PubMed1990

1 alpha,25-dihydroxyvitamin D3-induced increments in hepatocyte cytosolic calcium and lysophosphatidylinositol: inhibition by pertussis toxin and 1 beta,25-dihydroxyvitamin D3.

1 alpha,25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium and alters membrane phospholipid metabolism in hepatocytes. To define the causal relationship between these events, we examined the effects of 1 alpha,25-dihydroxyvitamin D3 on 32P-labeled lysophosphatidylinositol levels and cytosolic calcium as affected by pertussis toxin and 1 beta,25-dihydroxyvitamin D3, the biologically inactive analog. 32P-labeled lysophosphatidylinositol was determined by two-dimensional thin-layer chromatography. Cytosolic calcium was measured in cells loaded with quin-2AM. Within 5 min, 1 alpha,25-dihydroxyvitamin D3 increased hepatocyte cytosolic calcium by 31% (p less than 0.05) and 32P-labeled lysophosphatidylinositol by 38% (p less than 0.05). Pertussis toxin inhibited the hormone-induced rise in cytosolic calcium but not the increase in 32P-labeled lysophosphatidylinositol. Exposure to exogenous lysophosphatidylinositol for 5 min increased cytosolic calcium by 40% (p less than 0.05), an effect that was also inhibited by pertussis toxin. 1 beta,25-Dihydroxyvitamin D3 had no effect on either hepatocyte cytosolic calcium or 32P-labeled lysophosphatidylinositol but prevented the 1 alpha,25-dihydroxyvitamin D3-induced increments. The results suggest that a G protein sensitive to pertussis toxin is required for the transduction of the lysophosphatidylinositol signal but not the generation of the signal. The ability of 1 beta,25-dihydroxyvitamin D3 to inhibit the 1 alpha,25-dihydroxyvitamin D3-induced changes in phospholipids suggests that the epimer may compete with 1 alpha,25-dihydroxyvitamin D3 for an initiating receptor.

Animals↗
PubMed1990

1,25(OH)2D3 increases calcium and phosphatidylinositol metabolism in differentiating cultured human keratinocytes.

The effect of 1,25(OH)(2)D(3) on the intracellular calcium, (Ca(+2))i, in both cultured human keratinocytes and in cultured human dermal fibroblasts was investigated. When the intracellular calcium (Ca(+2))i in cultured human keratinocytes, grown in a serum-free medium containing 1.8 mM calcium, was measured by the fluorescent calcium-indicator, Furu-2, the (Ca(+2)i increased 154%, 202%, and 409% over the control value after incubation with 1,25(OH)(2)D(3) at 10(-10) m, 10(-8) m, and 10(-6) m, respectively. This response was immediate (15 seconds), specific (no effect with either 25(OH)D(3) at 10(-8) m or vitamin D(3) at 10(-8) m), and occurred with or without EGTA in the medium. In contrast, 1,25(OH)(2)D(3) did not increase the (Ca(2+))i in either cultured human keratinocytes that were grown in low calcium (0.05 mm), serum-free medium or in cultured human dermal fibroblasts that were grown in medium containing 0.05 mm calcium and 1% serum. The effect of 1,25(OH)(2)D(3) on the the turnover of phosphatidylinositol was investigated as a possible cause for the observed increase in (Ca(+2)i. Cultured human keratinocytes that were incubated with (3)H-inositol demonstrated a 50 % +/- 10% increase in the triphosphated, plasma membrane-bound metabolite of phosphatidylinositol, PIP(2), by 15 seconds, followed by a rapid decrease at 30 seconds, then a return toward basal levels by 1 minute. Lysophosphatidylinositol, which results from the sn-2 deacylation of phosphatidylinositol by phospholipase A(2), decreased 20% +/- 8% within 30 seconds, then increased to 200% +/- 10% of the control value by 5 minutes. The accumulation of IP(3) was increased 50% to 100% above the control value within 30 seconds and this increase was substained during the 5-minute incubation period. Stimulation of phosphatidylinositol turnover by 1,25(OH)(2)D(3) was not detected in either cultured human keratinocytes that were grown in serum-free, low calcium medium or in cultured human dermal fibroblasts that were grown in 1% serum.

Journal Article↗
PubMed1990

An evaluation of the relative contributions of exposure to sunlight and of diet to the circulating concentrations of 25-hydroxyvitamin D in an elderly nursing home population in Boston.

We investigated the vitamin D status of a Caucasian elderly population in a long-term-care facility in Boston. Comparison was made with a group of free-living elderly people. The sunlight exposure of residents was monitored and its effect on the serum concentrations of 25-hydroxyvitamin D [25(OH)D] was compared with contributions from diet and multivitamins. Seasonal changes in serum 25(OH)D concentrations caused by sunlight exposure were greatest in the free-living subjects and declined in magnitude as the mobility of the volunteers decreased. Diet failed to provide an adequate amount of vitamin D for volunteers who had minimal outdoor activity. Use of a multivitamin supplement containing 10 micrograms (400 IU) vitamin D maintained serum 25(OH)D concentrations greater than 37.5 nmol/L.

Activities of Daily Living↗
PubMed1990

The use and interpretation of assays for vitamin D and its metabolites.

Vitamin D is essential for the maintenance of calcium and bone metabolism in humans. The recommended daily allowance (RDA) for vitamin D in the United States of 200 IU (5.0 micrograms) is reasonable for adults who receive some exposure to sunlight; however, in the absence of any exposure to sunlight, this recommendation may be 2 to 3 times lower than that actually required to satisfy the body's needs. Vitamin D was first measured by bioassays. However, bioassays became obsolete in light of the revelation that vitamin D must be activated first in the liver to 25-hydroxyvitamin D (25-OH-D) and then in the kidney to 1,25-dihydroxyvitamin D [1,25(OH)2D] before becoming biologically functional. Current assays measure circulating concentrations of vitamin D, 25-OH-D or 1,25(OH)2D. The serum vitamin D concentration is of value for determining the role of sunlight in producing vitamin D in skin and as a provocative test to determine the absorption of vitamin D in patients with malabsorption syndromes. The serum concentration of 25-OH-D is most valuable for determining the overall vitamin D status of an individual, since it is an average of dietary and sunlight-induced vitamin D. The measurement of the serum 1,25(OH)2D concentrations has been most useful in evaluating disorders in calcium and bone metabolism related to acquired and inborn errors in the conversion of 25-OH-D to 1,25(OH)2D.

25-Hydroxyvitamin D 2↗
PubMed1989

Rapid actions of 1 alpha,25-dihydroxyvitamin D3 on Ca2+ and phospholipids in isolated rat liver nuclei.

The effects of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) on Ca2+ levels and phospholipid metabolism were studied in isolated nuclei prepared from rat liver. Nuclear Ca2+ concentration was estimated with the fluorescent indicator Fura 2. In agreement with previous reports, ATP (1 mM) produced a rapid increase in nuclear Ca2+ from 188 +/- 25 to 593 +/- 121 nM. Exposure to 1 alpha,25-(OH)2D3 (20 nM) also produced a rapid increase in nuclear Ca2+ to 402 +/- 71 nM. The 1 beta epimer of 1 alpha,25-(OH)2D3 had no effect. Nuclear phosphatidylinositol was labeled by incubation with [gamma-32P]ATP for 3 h. 1 alpha,25-(OH)2D3 produced a two-fold increase in [32P]lysophosphatidylinositol (LPI) within 5 min from 44 +/- 11 to 87 +/- 19 cpm/2.5 x 10(7) nuclei. 1 beta,25-(OH)2D3 had no effect on [32P]LPI production. Exposure of nuclei to exogenous LPI (15 microM) produced an instantaneous increase in nuclear Ca2+ to 372 +/- 81 nM, comparable to ATP and 1 alpha,25-(OH)2D3. The rapid effects of 1 alpha,25-(OH)2D3 on phospholipid metabolism and Ca2+ in isolated nuclei suggest that the steroid may exert effects distinct from the well-characterized receptor-mediated changes in gene expression.

Adenosine Triphosphate↗
PubMed1989

Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation.

Exposure to sunlight initiates the formation of vitamin D3 in skin as the UV B radiation in the solar spectrum causes the photoconversion of 7-dehydrocholesterol to previtamin D3. A heat-induced isomerization then converts previtamin D3 to vitamin D3 over a period of days. A number of irradiation products of vitamin D3 are known to form upon irradiation with high intensity UV radiation, but the effect of subsequent exposures to sunlight on the vitamin D3 formed in skin is not known. To investigate this phenomenon, human skin containing vitamin D3 was exposed to sunlight in Boston. A model system of [3H]vitamin D3 in methanol was also used to study the effects of sunlight on vitamin D3 throughout the year. Vitamin D3 proved to be exquisitely sensitive to sunlight, and once formed in the skin, exposure to sunlight resulted in its rapid photodegradation to a variety of photoproducts, including 5,6-transvitamin D3, suprasterol I, and suprasterol II.

Boston↗
PubMed1988

24-Dehydrovitamin D is potent inhibitor of rat liver microsomal vitamin D-25-hydroxylase.

A pathway has been described in the skin for the synthesis of 24-dehydrovitamin D3 (delta 24D3) from 24-dehydroprovitamin D3. The physiologic function of delta 24D3 is unknown, but has been proposed as a potential inhibitor of hepatic vitamin D-25-hydroxylase. We validated an assay for vitamin D-25-hydroxylase in rat hepatic microsomes, using nanomolar amounts of [3H]D3 as substrate, and found that delta 24D3 competitively inhibits vitamin D-25-hydroxylase activity. The apparent Ki was approximately 17 nM, indistinguishable from the Km of approximately 15 nM, suggesting that both delta 24D3 and cholecalciferol have similar affinity for the enzyme. We found no [3H]delta 24D3 in serum or liver extracts after repletion of vitamin D-depleted rats with [3H]vitamin D3 for 4 h or 6 days. A dose of 1 microgram delta 24D3 to vitamin D- and calcium-depleted rats was unable to promote any elevation in the 45Ca transport by everted duodenal sacs or to increase levels of plasma calcium: thus no evidence for biological conversion of delta 24D3 to vitamin D3 was observed. Further studies are needed to determine whether delta 24D3 is released from the skin to the circulation and is taken up by the liver, before physiological relevance can be attributed to this inhibitor.

Animals↗
PubMed1988

A novel approach for the evaluation and treatment of psoriasis. Oral or topical use of 1,25-dihydroxyvitamin D3 can be a safe and effective therapy for psoriasis.

We investigated the effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on cultured fibroblasts and keratinocytes from patients with psoriasis and treated 17 patients with psoriasis with orally or topically administered 1,25-(OH)2-D3. Cultured fibroblasts from three of five patients showed a normal response to the antiproliferative activity of a physiologic dose of 1,25-(OH)2-D3, whereas fibroblasts from the other two had a partial resistance to the drug. Cultured keratinocytes from two patients with psoriasis possessed nuclear receptors for 1,25-(OH)2-D3 and the drug caused a dose-dependent inhibition of proliferation and induction of terminal differentiation of these cells similar to effects in normal cultured keratinocytes. Ten of 14 patients with moderate to severe psoriasis who received oral 1,25-(OH)2-D3 showed significant clearing of their hyperkeratotic plaques. Three patients had complete clearing that was sustained with maintenance therapy, but four patients received little or no benefit from the therapy. By the administration of 1,25-(OH)2-D3 as a single oral dose at bedtime, larger doses of the drug could be tolerated without evidence of hypercalciuria or hypercalcemia. Three patients who received topical 1,25-(OH)2-D3 showed a rapid response with complete clearing after 6 weeks of therapy. Therefore, these preliminary findings suggest that orally or topically administered 1,25-(OH)2-D3 may be a safe and effective alternative therapy for the treatment of psoriasis.

Administration, Cutaneous↗
PubMed1988

Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes.

Exposure of the skin to sunlight results in both tanning and vitamin D3 production. It has therefore been suggested that vitamin D3 or its active metabolite 1,25-dihydroxyvitamin D3 may be the mediator of UV-induced melanogenesis. To test this hypothesis, newborn foreskin-derived melanocytes were cultured in paired dishes in hormone-supplemented medium with 2% serum containing no detectable vitamin D3 or in the same medium containing 10(-8) or 10(-10) M of either provitamin D3, lumisterol, previtamin D3, vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3. After 10 days, cell number in cultures containing vitamin D compounds was 93%-140% of unsupplemented controls and melanin content was 60%-120% of control, with no significant difference in either parameter for any compound tested. In separate experiments, human melanocytes and Cloudman S91 melanoma cells were repeatedly irradiated with physiologic doses of simulated sunlight and incubated between irradiations with provitamin D3, previtamin D3, vitamin D3, or 1,25-dihydroxyvitamin D3. Irradiated cultures had a 90%-95% inhibition of cell growth associated with a 200%-800% increase in melanin content per cell relative to controls, but there was no effect of any vitamin D compound on either cell type. Neither cultured human melanocytes nor S91 cells showed evidence of the cytosolic 1,25-dihydroxy-vitamin D3 receptor binding by sucrose density gradient analysis using radiolabeled 1,25-dihydroxyvitamin D3. The combined data strongly suggest that neither vitamin D3 nor its precursors or metabolites directly mediate melanogenesis in these cells.

Cells, Cultured↗