1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

Different effects of physiologically and pharmacologically increased growth hormone levels on cholecalciferol metabolism at prepubertal age

The aim of the study was to investigate the influence of physiologically and pharmacologically increased plasma growth hormone (GH) levels on cholecalciferol metabolism at prepubertal age. Three groups of dogs raised on the same diet were studied from weaning till 21 weeks of age, i.e., small breed dogs (n = 7, control group); large breed dogs with 15-fold greater growth rates compared to the control group (n = 8, LB-group); and small breed dogs treated with pharmacological doses of growth hormone (n = 6, GH-group; 0.5IU GH per kg body per day) from 12 to 21 weeks of age. Excess of GH had the expected anabolic effect on growth rate and phosphate sparing. Increased plasma GH levels in the LB- and GH-groups versus the control group were accompanied by (1) greater plasma insulin-like growth factor I (IGF-I) levels, (2) greater plasma 1,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) levels, and (3) lower plasma 24,25(OH)(2)D(3) levels. In the LB-group, excess of GH favored plasma 1,25(OH)(2)D(3) levels by decreasing the clearance of 1,25(OH)(2)D(3), whereas in the GH-group by increasing the production of 1,25(OH)(2)D(3). The lowered plasma 24,25(OH)(2)D(3) levels in the LB- and GH-groups were likely attributed to a competitive inhibition of the production of 24,25(OH)(2)D(3) by GH and/or IGF-I.     

1,25-Dihydroxyvitamin D(3) and 9-cis-retinoids are synergistic regulators of 24-hydroxylase activity in the rat and 1, 25-dihydroxyvitamin D(3) alters retinoic acid metabolism in vivo.

The RXR forms a heterodimer with the VDR to activate genes that are regulated by 1,25(OH)(2)D(3). In the absence of RXR's ligand, 9-cis-RA, RXR appears to be a silent partner to VDR. The effect of 9-cis-RA on VDR/RXR heterodimer formation and 1, 25(OH)(2)D(3)-mediated gene expression in vivo remains unclear. We examined the effect of exogenous 9-cis-RA or 9-cis-RA precursors, 9, 13-di-cis-RA and 9-cis-RCHO, on 1,25(OH)(2)D(3)-mediated induction rat renal 24-hydroxylase. The rats were treated as follows: (1) vehicle; (2) 1,25(OH)(2)D(3); (3) 1,25(OH)(2)D(3) + 9-cis-RA; (4) 1, 25(OH)(2)D(3) + 9,13-di-cis-RA; (5) 1,25(OH)(2)D(3) + 9-cis-RCHO; (6) 9-cis-RA; (7) 9,13-di-cis-RA; and (8) 9-cis-RCHO. 1, 25(OH)(2)D(3) was administered IP 18 h prior to sacrifice. The retinoids were administered every 4 h, starting 28 h prior to sacrifice. The last retinoid dose was administered 4 h prior to sacrifice. Treatment with 1,25(OH)(2)D(3) alone increased 24-hydroxylase from 35 +/- 6 (controls) to 258 +/- 44 pmol/min/g tissue. When 1,25(OH)(2)D(3) was administered with 9-cis-RA, 9, 13-di-cis-RA, or 9-cis-RCHO, 24-hydroxylases were 568 +/- 56, 524 +/- 56, and 463 +/- 62 pmol/min/g tissue, respectively. Furthermore, codosing of 1,25(OH)(2)D(3) and 9-cis-retinoids resulted in higher circulating concentrations of 9-cis-RA and 9,13-di-cis-RA when compared to rats dosed with 9-cis-retinoids alone. This was shown to be due to 1,25(OH)(2)D(3) increasing the half-life of 9,13-di-cis-RA by three to four times. These results show that 9-cis-RA can act synergistically with 1,25(OH)(2)D(3) in the regulation of 24-hydroxylase in vivo. Additionally, 1,25(OH)(2)D(3) regulates 9, 13-di-cis-RA metabolism in vivo.     

Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.     

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.     

Enhancing effects of ceramide derivatives on 1,25-dihydroxyvitamin D(3)-induced differentiation of human HL-60 leukemia cells

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] represents a useful approach for the treatment for cancer, including acute myeloid leukemia (AML). Recent studies demonstrated that the combined administration of 1,25-(OH)(2)D(3) and differentiation-enhancing agents could alleviate the side effects of 1,25-(OH)(2)D(3) and improve the rate of long term survival. In this study, we determined the enhancing activities of ceramide derivatives on 1,25-(OH)(2)D(3)-induced differentiation of human myeloid leukemia HL-60 cells. Importantly, some of these derivatives -- namely, A2, B3, and H9 -- enhanced the 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells in a concentration-dependent manner. In addition, the morphologic studies using Giemsa staining and flow cytometric analysis demonstrated that the combined treatment of 1,25-(OH)(2)D(3) with one of the three analogues, A2, B3, and H9, directed the HL-60 cells into monocytic lineage, but not into granulocytic lineage. The inhibition studies demonstrated that A2, B3, and H9, enhanced 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells via the PI3-K/PKC/JNK/ERK pathways. The ability of ceramide derivatives to enhance the differentiation-inducing potential of 1,25-(OH)(2)D(3) may contribute to an effective therapy for AML.     

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3)

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.     

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.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in normal rats. Serum (S) levels and urinary excretion of Ca2+ (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. The animals were housed in metabolic cages and 24-hr urine specimens were collected. After 24 hr SCa2+ increased similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3, while 24,25(OH)2D3 alone did not change SCa2+. UCaV after 24 hr increased significantly less (P less than 0.025) with 1,25(OH)2D3 + 24,25(OH)2D3 than with 1,25(OH)2D3 alone. After 5 days of 1,25(OH)2D3, SCa2+ rose from 5.1 +/- 0.15 to 6.29 +/- 0.08 whereas 1,25(OH)2D3 + 24,25(OH)2D3 effected a greater increase in SCa2+ up to 6.63 +/- 0.09 (P less than 0.01). 24,25(OH)2D3 alone did not change SCa2+. UCaV after 5 days of treatment rose similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3. After 10 days of 1,25(OH)2D3 SCa2+ was 6.17 +/- 0.15 meq/liter while with the combination SCa2+ rose to 6.74 +/- 0.2 (P less than 0.025). 24,25(OH)2D3 alone did not change SCa2+. These results show that (a) 24,25(OH)2D3 alone does not alter SCa2+ in normal rats, (b) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, and (c) it is suggested that the effect of 24,25(OH)2D3 on serum Ca2+ level, at least partly, may result from its hypocalciuric effect.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.     

Gene expression profiles in rat intestine identify pathways for 1,25-dihydroxyvitamin D(3) stimulated calcium absorption and clarify its immunomodulatory properties

Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).     

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.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

1,25-Dihydroxyvitamin D3 in lipiodol for the treatment of hepatocellular carcinoma: cellular, animal and clinical studies

1,25-Dihydroxyvitamin D(3) (1,25-(OH)(2) D(3)) is a potent regulator of cell growth and differentiation, with recent evidence showing inhibition of tumor invasion, angiogenesis and tumor cell death. The growth-inhibitory properties of 1,25-(OH)(2) D(3) could be harnessed in the treatment of patients with cancer if the development of systemic hypercalcemia is avoided. Hepatocellular cancer (HCC) presents a setting where the tumor is accessible for treatment through the hepatic artery and also where the tumor is highly lipiodol avid. On this basis, we hypothesised that, 1,25-(OH)(2) D(3) dissolved in lipiodol and administered through the hepatic artery may prove to be a rational approach to the use of the drug in the treatment of HCCs. In brief, 6 years of work with 1,25-(OH)(2) D(3) at cellular, animal and clinical level has provided us with plenty of support for this hypothesis. Sensitivity of HCCs in cell culture to 1,25-(OH)(2) D(3), growth retardation of human HCC xenografts in nude mice, uptake and retention of 1,25-(OH)(2) D(3)-lipiodol by liver tumors in cell culture and animals, escalation of the 1,25-(OH)(2) D(3) dose by 100x without the development of hypercalcemia in both liver tumor bearing rats and in patients with HCC are some of the evidence that will be discussed in this paper.