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Biomedical subjects

M F Holick

Publications and source records attributed to M F Holick.

At least 37 records · Page 2Linked to original sources
PubMed2000

Condition-dependent presence of beta-lipotropin-like peptide in human keratinocytes.

This study aimed to characterize the beta-endorphin-immunoreactive material (betaE-IR) detectable in normal human keratinocytes (NHK). The effects of different culturing conditions and UV-irradiation on production of betaE-IR by NHK were assessed by radioimmunoassay and HPLC. All culture systems contained low levels of betaE-IR that was increased in conditioned media after UV-irradiation under certain conditions. NHK grown in nutrient-poor medium contained highest levels of betaE-IR that exhibited beta-lipotropin-like properties after HPLC analysis. The other culturing conditions displayed no authentic betaE-related peptides. Our results indicate that under certain culturing conditions NHK can produce POMC peptides like beta-lipotropin, which can be induced by UV-radiation.

Cells, Cultured↗
PubMed2000

An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3.

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.

Calcitriol↗
PubMed2000

Immunoreactivity of six monoclonal antibodies directed against 1,25-dihydroxyvitamin-D3 receptors in human skin.

Using confocal laser scanning microscopy, we tested the suitability of five monoclonal mouse antibodies (IVA7E7, IVB12G12, IVG9C11, VD2F12, and VIIID8C12) that had been raised against different domains of the porcine intestinal 1,25-dihydroxyvitamin-D3 receptor (VDR), for the immunohistological detection of VDR in human skin. The VDR immunoreactivity of these antibodies was compared with the well-characterized VDR-staining pattern of the mouse monoclonal antibody 9A7gamma raised against chick intestinal VDR. All six antibodies revealed strong nuclear and qualitatively similar immunoreactivity in all cell layers of the viable epidermis. Our data demonstrate that the five mouse monoclonal antibodies are suitable for immunohistochemical detection of VDR in frozen sections. These antibodies show comparable staining patterns in human skin even though they had been raised against different functional domains of the 1,25-dihydroxyvitamin-D3 receptor.

Animals↗
PubMed2000

Is psoriasis a T-cell disease?

The etiology and pathogenesis of psoriasis--one of the most common chronic, inflammatory, hyperproliferative skin disorders of man--have long fascinated dermatologists, pathologists and biologists alike. Here, we have a model disease that offers to study neuroectodermal-mesenchymal interactions in the widest sense possible. Epithelial, endothelial, and hematopoietic cells as well as neurons projecting into the skin apparently all interact with each other to generate the characteristic psoriatic lesion. For decades, the ongoing controversy on the molecular nature, choreography and hierarchy of these complex interactions e.g. between epidermal keratinocytes, T cells, neurotrophils, endothelial cells and sensory nerves has served as a driving force propelling investigative dermatology to ever new horizons. This debate has not only been at the heart of our quest to develop more effective forms of therapy for this socially crippling disease, but it also has profoundly influenced how we view the skin as a whole: the numerous competing theories on the pathogenesis of psoriasis published so far also are reflections on the evolution of mainstream thought in skin biology over the last decades. These days, conventional wisdom infatuated with a T-cell-centered approach to inflammatory skin diseases-- portrays psoriasis as an autoimmune disease, where misguided T lymphocyte activities cause secondary epithelial abnormalities. And yet, as this CONTROVERSIES feature reminds us, some authoritative "pockets of academic resistance" are still quite alive, and interpret psoriasis e.g. as a genetically determined, abnormal epithelial response pattern to infectious and/or physicochemical skin insults. Weighing the corresponding lines of argumentation is not only an intriguing, clinically relevant intellectual exercise, but also serves as a wonderful instrument for questioning our own views of the skin universe and its patterns of deviation from a state of homeostasis.

Animals↗
PubMed2000

Hexafluoro-1,25-dihydroxyvitamin D3 has markedly increased potency in inhibiting proliferation of cultured human keratinocytes compared with 1,25-dihydroxyvitamin D3.

BACKGROUND: Although topical 1,25-dihydroxyvitamin D3 (calcitriol, 1, 25(OH)2D3) and its analogues, calcipotriol and tacalcitol, are effective for patients with psoriasis, some patients show little or no response. There is a need to develop more potent analogues of 1, 25(OH)2D3. Hexafluoro-1,25(OH)2D3 (F6-1,25(OH)2D3) is at least 10 times more potent than 1,25(OH)2D3 on calcium metabolism. OBJECTIVES: We were interested in whether F6-1,25(OH)2D3 was also more potent than 1,25(OH)2D3 in inhibiting normal human and psoriatic keratinocyte proliferation. METHODS: The antiproliferative activity of F6-1,25(OH)2D3 and 1,25(OH)2D3 was determined by 3H-thymidine incorporation into keratinocyte DNA and by counting basal cells. RESULTS: F6-1,25(OH)2D3 was approximately 10-fold more active and had a longer lasting antiproliferative effect than 1,25(OH)2D3 on normal human keratinocytes, and was about 100-fold more potent than 1, 25(OH)2D3 on human psoriatic keratinocytes as determined by 3H-thymidine incorporation. F6-1,25(OH)2D3 also caused a dose-dependent decrease in the number of basal cells and was 100-fold more active than 1,25(OH)2D3. CONCLUSIONS: The increased potency and the long-lasting effects of F6-1,25(OH)2D3 suggest that F6-1,25(OH)2D3 may be a potent candidate agent for treating psoriasis.

Calcitriol↗
PubMed2000

Expression of the circadian clock genes clock and period1 in human skin.

The circadian clock is a cellular machine composed of proteins with regulated expression that gives rise to circadian rhythms. Two main new concepts have arisen from recent research in the field in the last few years: (i) at least three to five key genes are involved in maintaining the basic circadian cellular rhythms, and (ii) their expression is fairly ubiquitous, extending beyond the traditionally considered pacemaker in mammals, the suprachiasmatic nucleus. We have demonstrated the expression of two circadian clock genes, clock and period1, in human skin cells. Reverse transcriptase polymerase chain reaction revealed the presence of clock and period1 mRNA in cultured human keratinocytes, melanocytes, and dermal fibroblasts, as well as in the human keratinocyte cell line HaCaT and the human melanoma line A375. In addition, antibodies to these two proteins produced immuno-positive staining in these cell types. Our investigations demonstrate for the first time that skin cells express circadian clock proteins constitutively although regulation of their expression and activity has not been elucidated. These proteins may have a role in cutaneous and/or systemic circadian biology and the skin and skin cells may provide an attractive model for the study of circadian rhythms.

CLOCK Proteins↗
PubMed2000

Decreased bioavailability of vitamin D in obesity.

BACKGROUND: Obesity is associated with vitamin D insufficiency and secondary hyperparathyroidism. OBJECTIVE: This study assessed whether obesity alters the cutaneous production of vitamin D(3) (cholecalciferol) or the intestinal absorption of vitamin D(2) (ergocalciferol). DESIGN: Healthy, white, obese [body mass index (BMI; in kg/m(2)) > or = 30] and matched lean control subjects (BMI </= 25) received either whole-body ultraviolet radiation or a pharmacologic dose of vitamin D(2) orally. RESULTS: Obese subjects had significantly lower basal 25-hydroxyvitamin D concentrations and higher parathyroid hormone concentrations than did age-matched control subjects. Evaluation of blood vitamin D(3) concentrations 24 h after whole-body irradiation showed that the incremental increase in vitamin D(3) was 57% lower in obese than in nonobese subjects. The content of the vitamin D(3) precursor 7-dehydrocholesterol in the skin of obese and nonobese subjects did not differ significantly between groups nor did its conversion to previtamin D(3) after irradiation in vitro. The obese and nonobese subjects received an oral dose of 50000 IU (1.25 mg) vitamin D(2). BMI was inversely correlated with serum vitamin D(3) concentrations after irradiation (r = -0.55, P: = 0.003) and with peak serum vitamin D(2) concentrations after vitamin D(2) intake (r = -0.56, P: = 0.007). CONCLUSIONS: Obesity-associated vitamin D insufficiency is likely due to the decreased bioavailability of vitamin D(3) from cutaneous and dietary sources because of its deposition in body fat compartments.

Adult↗
PubMed2000

A human skin equivalent model that mimics the photoproduction of vitamin D3 in human skin.

A human skin equivalent was prepared by culturing human keratinocytes on the surface of nylon filtration meshes containing human skin fibroblasts and by growing the epidermal cells at the air-liquid interface. This human skin equivalent model was used to mimic the photoproduction of vitamin D3 in human skin. It was found that the concentration of 7-dehydrocholesterol and its photoconversion to previtamin D3 and its subsequent thermal isomerization to vitamin D3 in the human skin equivalent was essentially identical to that of human skin. The 7-dehydrocholesterol content in the skin equivalent and human skin was 2187 +/- 296 and 2352 +/- 320 ng/cm2, respectively. The percentage of the major photoproducts of 7-dehydrocholesterol in the skin equivalent following ultraviolet B radiation (0.5 J/cm2) was 35% pre-vitamin D3, 29% lumisterol, and 6% tachysterol; 30% remained as 7-dehydrocholesterol. Similarly, in human skin they were 36%, 29%, 7%, and 28%, respectively. After incubation at 37 degrees C for 30 min, 11% and 12% of the previtamin D3 had thermally isomerized to vitamin D3 in the skin equivalent and human skin. In conclusion, compared with cultured keratinocytes or fibroblasts, the human skin equivalent model provides a superior in vitro system that better mimics the physiology and biochemistry of the photosynthesis of vitamin D3 in human skin.

Cholecalciferol↗
PubMed2000

Transient osteoporosis of hip in pregnancy.

Transient osteoporosis in pregnancy is a relatively rare disorder characterized by disabling pain in the hip without prior trauma and by striking radiographic evidence of osteopenia that is limited to the hip. Clinical and radiographic findings regress spontaneously within months after pregnancy and without late sequelae. In this study, we report on a case of a 28-yr-old Caucasian woman who suffered from transient osteoporosis during pregnancy. Nine months later, the patient was asymptomatic and there was resolution of the bone marrow edema pattern seen on the initial magnetic resonance imaging scan of her hip. Follow-up bone densitometry at 1 yr revealed a dramatic increase (12.3%) in bone mineral density of the symptomatic hip. We review the literature and discuss possible mechanisms involved in the pathogenesis of transient osteoporosis in pregnancy.

Absorptiometry, Photon↗
PubMed2000

Metabolism of [3alpha-3H] 25-hydroxyvitamin D2 in kidneys isolated from normal and vitamin D2-intoxicated rats.

With the availability of A-ring labelled 25OHD2, [3alpha-3H] 25OHD2, we have performed the present study to examine the metabolism of 25OHD2 using physiological substrate concentrations in perfused kidneys isolated from both normal and vitamin D2-intoxicated rats. Our results indicate that [3alpha-3H] 25OHD2 is metabolized into both 24(S),25,28-trihydroxyvitamin D2 [24(S),25,28(OH)3D2] and 24(R),25,26-trihydroxyvitamin D2 [24(R), 25,26(OH)3D2], and the amounts of these two metabolites produced in the kidney of vitamin D2-intoxicated rat were about 3-5 times higher than those produced in the kidney of normal rat. Similar results were also obtained with rat kidney homogenates incubated with [3alpha-3H] 25OHD2. Furthermore, we noted that the production of both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 in the kidney homogenates of vitamin D2-intoxicated rats increased with the time of incubation and then subsequently decreased. The decrease in both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 coincided with an increase in the fraction of total radioactivity distributed in the aqueous phase of the kidney homogenates. This finding suggested the possibility of further metabolism of 24(S),25,28(OH)3D2 and 24(R), 25,26(OH)3D2 into polar water-soluble metabolite(s). We then measured the radioactivity in the aqueous phase of kidney homogenates of both normal and vitamin D2-intoxicated rats incubated with [3alpha-3H] 25OHD2. It was noted that the amount of radioactivity in the aqueous phase of kidney homogenates of vitamin D2-intoxicated rats is higher than that present in the aqueous phase of kidney homogenates of normal rats. Thus, our study provides evidence for the first time for the formation of both 24(S),25,28(OH)3D2 and 24(R),25, 26(OH)3D2 under physiological conditions, and the possibility of their further metabolism into as yet unidentified polar water-soluble metabolite(s). As the formation of all these metabolites is increased in the kidney of vitamin D2-intoxicated rats when compared to normal rats, it appears that the increased rate of metabolism of 25OHD2 during hypervitaminosis D2 plays a significant role in the deactivation of 25OHD2.

25-Hydroxyvitamin D 2↗
PubMed2000

The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer.

Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25-dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3-1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10(-9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer.

Calcifediol↗
PubMed2000

Calcium and vitamin D. Diagnostics and therapeutics.

Vitamin D is neither a vitamin nor a nutrient if adequate exposure to sunlight is available to produce adequate quantities of vitamin D3 in the skin. It is well known that an adequate supply of vitamin D, either from the diet or from the skin, is important for maximum bone health throughout life. The new revelation that 25(OH)D can be metabolized to 1,25(OH)2D in the colon, prostate, and skin opens a new chapter in the vitamin D story. It is quite possible that there are two levels of vitamin D sufficiency. One level requires that the serum 25(OH)D levels be at least 20 ng/mL to satisfy the body's requirement for the renal production of 1,25(OH)2D that regulates calcium absorption, and bone calcium mobilization and bone mineralization. The second level may need higher circulating levels of 25(OH)D for maximum cellular health because of the conversion of 25(OH)D to 1,25(OH)2D in extrarenal tissues, such as the prostate, colon, and skin.

Adult↗
PubMed1999

An immunocytochemical approach to the study of beta-endorphin production in human keratinocytes using confocal microscopy.

Proopiomelanocortin (POMC) is a protein that is posttranslationally processed to yield POMC peptides. The main site of POMC expression is the anterior pituitary lobe but many other sources have been identified. There is evidence that the skin produces POMC peptides, although their roles have not yet been defined. In the skin, regulation of POMC gene expression is known to be hair-cycle dependent, and it is localized to the sebaceous gland. In particular, beta-endorphin, a POMC peptide, has been shown to be modulated by TPA, IL-1 alpha, and ultraviolet radiation in keratinocytes. These results were obtained by examination of POMC mRNA levels using the Northern blot method; beta-endorphin protein production by the Western blot method on cultured cells; and immunocytochemistry for tissue preparations. This report represents an approach to use immunocytochemistry to quantify beta-endorphin production in cultured human keratinocytes. Additionally, we examined whether exposure to 20 mJ ultraviolet B radiation (UVB) and/or UVA could influence beta-endorphin production in these cells. Keratinocytes were grown in monolayers, in serum-free medium, fixed, and incubated with antiserum to whole synthetic beta-endorphin. Fluorescence microscopy was performed with a confocal laser scanning microscope. The integrated level of fluorescence was evaluated in n = 18 +/- 8 individual cells, and this was assumed to be proportional to beta-endorphin content. High variability was observed in the fluorescence intensity among cells. No significant differences between control and UVB- or UVA + UVB-treated cells was found. Similar results were produced by using brefeldin A, a compound that disrupts the secretory pathway, eliminating the possibility that the absence of a difference between beta-endorphin content in the treated and control cells was due to secretion of the peptide into the medium. We conclude that: (1) beta-endorphin or beta-endorphin-like peptides are produced in human keratinocytes and are readily detected by immunocytochemistry; (2) under the conditions tested, UVA and/or UVB did not increase beta-endorphin-like immunoreactivity in these cells.

Cells, Cultured↗
PubMed1999

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.

3T3 Cells↗
PubMed1999

Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1alpha-hydroxylase gene.

The human 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene has been cloned. It contained nine exons and eight introns spanning approximately 6.5 kb and a 1.4-kb 5'-flanking region. The 5'-flanking region contains consensus or highly conserved sequences for TATA, Pu, and CCAAT boxes, four cAMP response elements, two activator protein-1 (AP-1) response elements, two AP-2 response elements, three specific protein-1 (Sp1) response elements, and four NF-kappaB binding sites, but no vitamin D response element. By using luciferase reporter gene constructs of truncated forms of the 1alpha-OHase promoter transfected into a modified pig kidney cell line, AOK-B50, we identified regulatory regions of the 1.4-kb 1alpha-OHase promoter for parathyroid hormone 1-34 [PTH(1-34)], forskolin, and 1,25-hydroxyvitamin D3 [1,25(OH)2D3]. The 1.4-kb 1alpha-OHase promoter (AN1) modestly (1.7-fold) induced luciferase activity, whereas 1,100- (AN2), 827- (AN3), 672- (AN4), 463-(AN5), and 363-bp (AN6)-truncated promoters greatly stimulated luciferase activity by 494-fold, 18.4-fold, 55.3-fold, 643-fold, and 56.4-fold, respectively. PTH(1-34) and forskolin stimulated the activity of all constructs to varying degrees with significantly greater responsiveness for both compounds on AN2 and AN5. 1,25(OH)2D3 suppressed PTH(1-34)-induced activity on AN2 and AN5 constructs by 58% and 52%, respectively, but had no effect on the other constructs. These studies characterize the regulatory regions of the human 1alpha-OHase gene and provide insight into the physiologic basis for regulation of the expression of this gene by PTH and 1,25(OH)2D3.

Base Sequence↗
PubMed1999

A liposomal model that mimics the cutaneous production of vitamin D3. Studies of the mechanism of the membrane-enhanced thermal isomerization of previtamin D3 to vitamin D3.

We reported previously that the rate of previtamin D3 (preD3) <==> vitamin D3 isomerization was enhanced by about 10 times in the skin compared with that in organic solvents. To elucidate the mechanism by which the rate of this reaction is enhanced in the skin, we developed a liposomal model that mimicked the enhanced isomerization of preD3 to vitamin D3 that was described in human skin. Using this model we studied the effect of changing the polarity of preD3 as well as changing the chain length and the degree of saturation of liposomal phospholipids on the kinetics of preD3 <==> vitamin D3 isomerization. We found that a decrease in the hydrophilic interaction of the preD3 with liposomal phospholipids by an esterification of the 3beta-hydroxy of preD3 (previtamin D3-3beta-acetate) reduced the rate of the isomerization by 67%. The addition of a hydroxyl on C-25 of the hydrophobic side chain (25-hydroxyprevitamin D3), which decreased the hydrophobic interaction of preD3 with the phospholipids, reduced the rate by 87%. In contrast, in an isotropic n-hexane solution, there was little difference among the rates of the conversion of preD3, its 3beta-acetate, and 25-hydroxy derivatives to their corresponding vitamin D3 compounds. We also determined rate constants (k) of preD3 <==> vitamin D3 isomerization in liposomes containing phosphatidylcholines with different carbon chain lengths. The rates of the reaction were found to be enhanced as the number of carbons (Cn) in the hydrocarbon chain of the phospholipids increased from 10 to 18. In conclusion, these results support our hypothesis that amphipathic interactions between preD3 and membrane phospholipids stabilize preD3 in its "cholesterol like" cZc-conformer, the only conformer of preD3 that can convert to vitamin D3. The stronger these interactions were, the more preD3 was likely in its cZc conformation at any moment and the faster was the rate of its conversion to vitamin D3.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

An international comparison of serum 25-hydroxyvitamin D measurements.

Vitamin D status is usually assessed by measuring the serum 25-hydroxyvitamin D (25(OH)D) concentration. This mainly depends on sunshine exposure, nutrition and age. Interlaboratory variation may hamper comparison between results from different populations. This study reports cross-calibration of the 25(OH)D assays of five laboratories. In study 1, serum 25(OH)D was measured with three different assays in 104 serum samples from a large vitamin D supplementation study. The mean serum 25(OH)D level was 80% higher when measured by competitive protein binding (CPB) assay than by high-performance liquid chromatography (HPLC), while radioimmunoassay (RIA) gave intermediate values. The highest correlation was observed between RIA and HPLC (r = 0.84, p <0.01). Of the serum 25(OH)D values in the lowest quartile by HPLC, 25% were not recognized by CPB and 21% were not recognized by RIA as belonging to the lowest quartile. In study 2, the five laboratories analyzed serum 25(OH)D in eight serum samples covering the concentration range very low to high, with five different assays. The differences between the mean values for serum 25(OH)D between the laboratories with the highest and lowest values was 38%. The ranking order of individual samples according to the serum 25(OH)D value was very similar in all laboratories. The results show that 25(OH)D values from different laboratories can not be assumed to be comparable unless a careful cross-calibration has been performed.

Aged↗