Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

A simple method for the isolation of vitamin D metabolites from plasma extracts

A simple method has been developed using 'SEP-PAK' disposable silica cartridges to separate the major endogenous vitamin D metabolites, namely vitamin D3, 25-hydroxy vitamin D3 (25OHD3), 1,25 dihydroxy vitamin D3 (1.25 (OH)2D3) and 24,25 dihydroxyvitamin D3 (24,25 (OH) 2D3). After extraction of plasma in isopropanol-toluene (25:75) the dried extract is reconstituted in hexane; this is applied to a SEP-PAK column, and stepwise elution carried out under gravity with 0.1 divided by isopropanol in hexane (neutral lipids), 1% isopropanol in hexane (D3), 3 divided by isopropanol in hexane (25OHD3), 3.125 divided by ethanol in dichloromethane (24,25 (OH) 2D3) and 50 divided ethanol in toluene (1, 25(OH) 2D3). Complete separation of these D3 metabolites is achieved by this process and up to 40 samples can be handled at one time.If combined with a suitable ligand binding assay, the system appears to be suitable for preparation of samples prior to the routine assay of vitamin D metabolites.     

Seasonal fluctuations in serum concentrations of vitamin D metabolites in normal subjects.

Serum concentrations of 25-hydroxycholecalciferol (25-OHD), 24,25-dihydroxycholecalciferol (24,25-(OH)2D), and 1,25-dihydroxycholecalciferol (1,25-(OH)2D) were measured at monthly intervals throughout the year in eight normal subjects. 25-OHD was measured by competitive protein-binding assay after Sephadex LH 20 chromatography, 24,25-(OH)2D by competitive protein-binding assay after Sephadex LH 20 and high-pressure chromatography, and 1,25-(OH)2D by radioimmunoassay after the same separation procedure as for 24,25-(OH)2D. A seasonal variation, apparently dependent on exposure to ultraviolet light, was found for all three metabolites. A study in six other normal subjects showed that there was no diurnal rhythm in any of the metabolites. Oral administration of 2 microgram 1,25-(OH)2D caused a sharp rise in serum concentrations of 1,25-(OH)2D and no change in the concentrations of the two other metabolites, but by 12 hours the 1,25-(OH)2D concentration had returned to the basal value. The concentrations of all three metabolites studied vary according to the season. Thus to interpret these concentrations in any subject the normal range for the particular season must be referred to.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Vitamin D3 metabolites in rat epididymis: high 24,25-dihydroxy vitamin D3 levels in the cauda region

Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D3 ([3H]25(OH)D3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [3H]-25(OH)D3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D3 and the 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH)2D3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH)2D3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in rat reproductive tissues.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

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.     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

Developmental changes in responsiveness to vitamin D metabolites

We have demonstrated that epiphyseal chondroblasts contain specific receptors for 24R,25-dihydroxy vitamin D3(24,25(OH)2D3) while diaphyseal osteoblasts contain specific receptors for 1 alpha 25-dihydroxy vitamin D3(1,25(OH)2D3). Both metabolites induce DNA synthesis and creatine kinase (CKBB) activity. We have also found that the responsiveness of rat kidney to these metabolites changes during development. In embryonic and early postnatal stages, the kidney responds to 24,25(OH)2D3, later to both 24,25(OH)2D3 and 1,25(OH)2D3, and the mature kidney only to 1,25(OH)2D3. These responses correlate with changes in the specific receptors present in the kidney. Furthermore, we have compared developmental changes in skeletal (epiphysis, diaphysis and mandibular condyle) and non-skeletal (kidney, cerebellum, cerebrum, liver and pituitary) tissue in both rat (a postnatal developer) and rabbit (a perinatal developer). Epiphyseal or diaphyseal chondroblasts at any stage of development were predominantly responsive to 24,25(OH)2D3, whereas osteoblasts were responsive to 1,25(OH)2D3. In contrast, condylar chondroblasts, kidney, cerebellum and pituitary responded to 24,25(OH)2D3 during early development and subsequently developed responsiveness to 1,25(OH)2D3. Using primary cell cultures from kidneys at different stages of maturation, we showed the same developmental pattern as in vivo. Chronic treatment of the cells with 24,25(OH)2D3, but not 1,25(OH)2D3, caused precocious development of responsiveness to 1,25(OH)2D3 in culture. We suggest that 24,25(OH)2D3 acts as a maturation factor, during early development in kidney, and probably in other tissues, possibly by induction of receptor to 1,25(OH)2D3, accompanied by down-regulation of its own receptor.     

Induction of 25-OH-vitamin D3 24- and 23-hydroxylase activities in partially purified renal extracts from pigs given exogenous 1,25-(OH)2D3

A renal mitochondrial cytochrome P 450 preparation from pigs treated with exogenous 1,25-(OH)2D3 was reconstituted with an NADPH-generating system, adrenodoxin and adrenodoxin reductase. The reconstituted system catalyzed the conversion of the substrate, 25-OH-D3, to metabolites comigrating with authentic 23,25-(OH)2D3 and 24,25-(OH)2D3 in both straight- and reverse-phase high-performance liquid chromatography systems, which achieve separation of these metabolites from each other as well as from other vitamin D metabolites. The putative 23,25-(OH)2D3 product was resistant to periodate treatment, while the 24,25-(OH)2D3 product was sensitive, providing additional evidence for the identity of the products. Although induction of 24-hydroxylase activity has been studied using renal homogenates from several species, only recently have techniques become available to study the activity of the enzyme in a solubilized and reconstituted form. Using these techniques, the present study shows that production of 24,25-(OH)2D3 was increased more than 80-fold with 1,25-(OH)2D3 treatment compared with untreated controls, an effect much greater than that previously observed with homogenates. In addition, production of both 23,25-(OH)2D3 and 24,25-(OH)2D3 varied with substrate concentration and was consistent with a monooxygenase-linked enzyme reaction.     

Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.     

1,25-Dihydroxyvitamin D3 binds specifically to rat vascular smooth muscle cells and stimulates their proliferation in vitro

Cultured vascular smooth muscle cells (VSMC) derived from rat aorta were found to contain a specific receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Its Kd (5.0 x 10(-11) M) and capacity (22.9 fmol/mg of cytosol protein) for 1,25-(OH)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D3 metabolites [1,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than vitamin D3] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(OH)2D3 receptor in other tissues. 1,25-(OH)2D3 at concentrations of more than 10(-10) M caused dose-dependent enhancement of the proliferation of VSMC in DMEM with 10% FCS. 25-Hydroxyvitamin D3 stimulated the proliferation of VSMC only at its highest concentration tested (10(-6) M). These data show that 1,25-(OH)2D3 stimulates the proliferation of VSMC after its binding to a cytoplasmic receptor of the cells in vitro, and support the possibility that VSMC are target cells of the hormone.     

Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals

Chromatographic methods suitable for the resolution of 24,25-dihydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 25-hydroxyvitamin D₃-26,23 lactone, and 25,26-dihydroxyvitamin D₂ are described. These four metabolites comigrated in high-pressure liquid chromatography on silicic acid columns developed in 11:89 isopropanol:hexane. Adequate resolution was achieved by subjecting the four-metabolite complex to high-pressure liquid chromatography column developed in 2:98 isopropanol:methylene chloride. This additional chromatographic step, coupled with modifications of assay procedures previously described, allowed for the estimation of plasma concentrations of vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 24,25-dihydroxyvitamin D₃, 25,26 dihydroxyvitamin D₂, 25,26-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃-26,23 lactone, and 1,25-dihydroxyvitamin D (1,25-dihydroxyvitamin D₂ plus 1,25-dihydroxyvitamin D₃). The samples automatically were introduced onto the high-pressure liquid chromatography columns with a Waters 710A “intelligent” processor. The metabolites were automatically collected with the aid of a programmable timer that advanced a fraction collector at predetermined intervals. The assays were used to determine the plasma vitamin D and vitamin D metabolite concentrations in five species of adult farm animals.     

24,25(OH)2D3 attenuates the calcemic effect of 1,25(OH)2D3 in rats with reduced renal mass

The present study was undertaken to evaluate the effect of 24,25(OH)2D3 on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)2D3, (iii) rats treated with 24,25(OH)2D3, and (iv) rats treated with 1,25(OH)2D3 and 24,25(OH)2D3. After 4 days, serum calcium in the 1,25(OH)2D3-treated group was 7.13 +/- 0.32 meq/liter (P less than 0.001 vs control). With the combination of 1,25(OH)2D3 and 24,25(OH)2D3 serum calcium was higher than that in control, 6.25 +/- 0.5 meq/liter (P less than 0.001 vs control), but lower than that in rats receiving 1,25(OH)2D3 alone (P less than 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)2D3 alone. On the eighth day serum calcium in the 1,25(OH)2D3-treated group, 6.52 +/- 0.25, was higher than in the 1,25(OH)2D3 + 24,25(OH)2D3 group, 5.87 +/- 0.17 meq/liter, P less than 0.05, P less than 0.001 vs control. In both 1,25(OH)2D3- and 1,25(OH)2D3 + 24,25(OH)2D3-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)2D3-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)2D3 and 24,25(OH)2D3 attenuates the calcemic response to 1,25(OH)2D3 without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)2D3 on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)2D3 on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)2D3 on the bone.     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

25-hydroxy-vitamin d metabolism in human colon cancer cells during tumor progression

RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1alpha-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH)3-D3 and 1,25-(OH)2- 24-oxo-D3. Likewise, 25-(OH)-D3 is metabolized into 24,25-(OH)2D3, 23,25-(OH)2D3, and 23,25-(OH)2-24-oxo-D3. Poorly-differentiated cancers expressed low levels of 1alpha-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1alpha,25-(OH)2-D3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1alpha,25-(OH)2-D3 particularly at late stages of colon cancer progression.     

Regulation of parathyroid hypertensive factor secretion by vitamin D3 analogs in parathyroid cells derived from spontaneously hypertensive rats

Parathyroid hypertensive factor (PHF) is a novel substance secreted by the parathyroid gland (PTG), which is elevated in 30-40% of all hypertensive patients; specifically, the low-renin subset. However, very little is known about the regulation of PHF secretion. Since the classical parathyroid regulator, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), may be elevated concurrent with or preceding the development of low-renin hypertension and elevated plasma PHF, we hypothesized that 1,25-(OH)2D3 would stimulate PHF release. To test this hypothesis, PTG organ and cell cultures, derived from spontaneously hypertensive rats (SHR) and the normotensive genetic control Wistar Kyoto (WKY) rats, were exposed to various vitamin D3 metabolites and PHF release measured by ELISA. 1,25-(OH)2D3 rapidly stimulated PHF release with enhanced sensitivity in SHR versus WKY cultures indicated by a leftward shift in the dose-response curve, whereas 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) had the converse effect. Vitamin D3 analog "BT," an agonist for the classical nuclear vitamin D receptor (1,25VDR(nuc)), was without effect suggesting a 1,25VDR(nuc)-independent mechanism and potential involvement of the plasma membrane-bound vitamin D receptor (1,25 D3-MARRS). Interestingly, protein expression of the 1,25 D3-MARRS was increased in SHR versus WKY parathyroid cells. In conclusion, these results support the idea that 1,25-(OH)2D3 may contribute to elevated plasma PHF in the SHR.     

Calcitonin secretion during postnatal development in the rat: beta-adrenergic agents and vitamin D3 metabolites

The possible contribution of catecholamines and vitamin D3 metabolites to the high plasma calcitonin (CT) levels in suckling baby rats is unknown. So, in vivo and in vitro (using a perifusion system) effects of beta-adrenergic agents and vitamin D3 metabolites on CT release were studied in the rat during the postnatal development. In 13-day-old rats, the increase in plasma CT levels induced by isoproterenol injection (0.1 micrograms/kg b.w.) was inhibited by a previous administration of propranolol. A significant decrease in plasma CT levels was observed after propranolol injection in baby rats (0.68 +/- 0.05 ng/ml vs. 0.93 +/- 0.01 ng/ml). A daily injection of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3; 25 pmoles/rat/day during 4 days) induced a marked rise in plasma calcium (16.1 +/- 0.2 mg/dl), and a great decrease in thyroidal CT contents (approximately 70% of control values) in 13-day-old rats while no change was noted with 24,25-dihydroxycholecalciferol (24,25-(OH)2D3). A negative correlation between plasma calcium and thyroidal CT stores was found in suckling and in weaning rats treated with different doses of 1,25-(OH)2D3, suggesting an indirect effect of 1,25-(OH)2D3 on CT secretion. The mobilization of the thyroidal CT content was greater in weaning than in suckling rats in response to a given hypercalcemia. In vitro, 5 X 10(-5) M isoproterenol induced a rapid increase in CT secretion rate while 1,25-(OH)2D3 inhibited the rise in CT release induced by 3.0 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)