Of 1 Alpha Research Articles

The Association between Alfacalcidol (1-alpha-hydroxyvitamin D3) and Oxidative Stress in Patients with Type II Diabetic Nephropathy

Diabetic nephropathy is the major risk factor for cardiovascular disease among young adults with DM Type (II), one of the more common microvascular complications, which influence the survival and quality of life of the patient. Research currently is underway to establish if there is a link between the effect of 1-alpha (OH) D3 and Type II Diabetic Nephropathy patients and whether the Type II regulated diabetic nephropathy patient is related to 1-alpha (OH) D3 and to oxidative stress conditions. In diabetic patients, no relationship has been established between the effect of alfacalcidol and antioxidant and/or prooxidant. The clinical applicability of our research will be to identify a correlation between diabetic nephropathy and oxidative stress. This will lead to the development of new guidelines to assess if this vitamin can be substituted or supplemented to treat this high mortality disease in patients with type II diabetes mellitus.

Calcitonin, a Regulator of the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene

Although parathyroid hormone (PTH) induces 25-hydroxyvitamin D(3) (25(OH)D(3)) 1alpha-hydroxylase (1alpha(OH)ase) under hypocalcemic conditions, previous studies showed that calcitonin, not PTH, has an important role in the maintenance of serum 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) under normocalcemic conditions. In this study we report that 1alpha(OH)ase transcription is strongly induced by calcitonin in kidney cells and indicate mechanisms that underlie this regulation. The transcription factor C/EBPbeta is up-regulated by calcitonin in kidney cells and results in a significant enhancement of calcitonin induction of 1alpha(OH)ase transcription and protein expression. Mutation constructs of the 1alpha(OH)ase promoter demonstrate the importance of the C/EBPbeta binding site at -79/-73 for activation of the 1alpha(OH)ase promoter by calcitonin. The SWI/SNF chromatin remodeling complex was found to cooperate with calcitonin in the regulation of 1alpha(OH)ase. Chromatin immunoprecipitation analysis showed that calcitonin recruits C/EBPbeta to the 1alpha(OH)ase promoter, and Re-chromatin immunoprecipitation analysis (sequential chromatin immunoprecipitations using different antibodies) showed that C/EBPbeta and BRG1, an ATPase that is a component of the SWI/SNF complex, bind simultaneously to the 1alpha(OH)ase promoter. These findings are the first to address the dynamics between calcitonin, C/EBPbeta, and SWI/SNF in the regulation of 1alpha(OH)ase and provide a mechanism, for the first time, for calcitonin induction of 1alpha(OH)ase. Because plasma calcitonin as well as 1,25(OH)(2)D(3) have been reported to be increased during pregnancy and lactation and in early development, these findings suggest a mechanism that may account, at least in part, for the increase in plasma 1,25(OH)(2)D(3) during these times of increased calcium requirement.

Insufficient renal 1-alpha hydroxylase and bone homeostasis in aged rats with insulin resistance or type 2 diabetes mellitus

This study aimed to explore the relationship between insufficient renal 1-alpha hydroxylase (IRH) and bone homeostasis in type 2 diabetes mellitus (T2DM) or insulin resistance (IR) and to investigate whether IR plays a major role in the pathogenesis of both IRH and bone loss in T2DM. The experimental animal models of T2DM, IR, IR treated with vitamin D (VD), IR treated with 1-alpha hydroxyvitamin D (1alpha(OH) D, the product of renal 1-alpha hydroxylase), T2DM treated with VD, and T2DM treated with 1alpha(OH) D were established on 18-month-old male Wistar rats. For rats in each animal model and normal control rats, IR was detected by euglycemic insulin clamp technique (EICT) and glucose infusion rate (GIR, an index of IR) was calculated. Levels of serum 25-hydroxyvitamin D (25(OH)D) and serum active vitamin D (1,25(OH)(2)D) were determined by radioimmunoassay (RIA), and 1,25(OH)(2)D/25(OH)D ratio (1,25-25-R, an index of renal 1-alpha hydroxylase activity in vivo) was calculated; and bone mineral density (BMD) in femoral bone and lumbar vertebrae was measured by dual-energy X-ray absorption (DEXA). No significant difference was observed among the levels of 25(OH)D in all the rats. In IR rats, 1,25(OH)(2)D level, 1,25-25-R, and BMD level were significantly higher than those in T2DM rats and were lower than those in normal control rats. In the aged rats with T2DM or IR, administration of VD had no effect on 25(OH)D level, 1,25(OH)(2)D level, 1,25-25-R, and BMD level. Administration of 1alpha(OH) D had also no effect on 25(OH)D level but increased 1,25(OH)(2)D level, 1,25-25-R, and BMD level. For the aged rats with T2DM or IR, GIR positively correlated with both levels of 1,25(OH)(2)D and BMD, and 1,25-25-R positively and significantly correlated with levels of BMD. In T2DM or IR, IRH is a precipitating factor for bone loss. IR seems to play a major role in the pathogenesis of both IRH and bone loss in T2DM.

Hereditary Vitamin D-resistant Rickets in Greek Children: Genotype, Phenotype, and Long-term Response to Treatment

We describe four patients (two pairs of children from two unrelated kindreds) from a Greek island, suffering from hereditary vitamin D-resistant rickets (HVDRR) with alopecia. There were two different homozygous mutations in the vitamin D receptor (VDR) gene of the affected members of the two kindreds that resulted in a truncated or missing receptor. The disorder began in early infancy with similar clinical, biochemical and radiological findings in all four patients, namely, alopecia (which provided the initial diagnostic evidence for HVDRR), rachitic deformities, hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum levels of 1,25-dihydroxyvitamin D; however, the patients of kindred B had a more severe clinical expression. Treatment options include oral or intravenous calcium and active vitamin D metabolites. The response varies widely in different cases. Our patients were initially treated with high doses of 1alpha(OH)D3 and oral calcium supplementation. Kindred A patients had a satisfactory response to this regimen, while kindred B patients presented clinical and biochemical improvement when 1alpha(OH)D3 was changed to 1,25(OH)2D3. In the older patients of each kindred, treatment requirements gradually decreased during puberty, and therapy was finally discontinued before adulthood.

Overexpression of ER and VDR is not sufficient to make ER-negative MDA-MB231 breast cancer cells responsive to 1 -hydroxyvitamin D5

1alpha-hydroxyvitamin D(5) [1alpha(OH)D(5)] is an active vitamin D analog showing promising chemopreventive effect in breast carcinogenesis. We previously reported that estrogen receptor (ER)-positive breast cancer cells were sensitive, whereas ER-negative breast cancer cells were relatively resistant to their antiproliferative effects. In the present study, we used ER-negative MDA-MB231, ER-transfected MDA-MB231 (S30) and ER-positive BT474 cell lines to evaluate the possible association between ER status and cellular sensitivity to 1alpha(OH)D(5) treatment. Our results demonstrate that ER expression in ER-negative breast cancer cells (S30) did not increase the sensitivity to 1alpha(OH)D(5), whereas in ER-positive BT474 cells, the significant antiproliferative effect of 1alpha(OH)D(5) was correlated with the downregulation of ER and progesterone receptor expression. Further analysis indicated that both MDA-MB231 and S30 cells express low vitamin D receptor (VDR) at transcriptional level and protein level. However, transfection of VDR failed to restore the sensitivity to 1alpha(OH)D(5) in MDA-MB231 and S30 cells, although VDR direct target gene CYP24 was more responsive to 1alpha(OH)D(5) treatment in MDA-MB231 and S30 cells overexpressing VDR. In addition, nuclear receptor cofactors NCoR1 and SRC1 that could potentially affect VDR action were also low in both MDA-MB231 and S30 cells in comparison with ER-positive, vitamin D-sensitive BT474 cells. These results suggest that in addition to the increased ER and VDR expression, the intact VDR signaling machinery as present in ER-positive, vitamin D-sensitive cells is essential for the antiproliferative action of vitamin D, whereas the direct VDR target genes such as CYP24 can remain responsive to augmented VDR expression.

Trace Determination of Steroids Causing Age-Related Diseases Using LC/MS Combined with Detection-Oriented Derivatization

With the rapid shift to an aging society in Japan, age-related diseases, such as osteoporosis, dementia and cancer, are sharply increasing. The measurement of steroids related to these diseases in biological fluids and tissues is useful for elucidation of the nature, diagnosis and treatment of such diseases. LC/MS is considered to be the most promising method for this purpose due to its specificity and versatility, but it sometimes does not demonstrate the required sensitivity for trace amounts of steroids, because steroids have a rather low response using either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). To overcome this problem, the author developed detection-oriented derivatization procedures for steroids in LC/MS. For ESI-MS, introduction of a permanently charged moiety is effective. Based on this, 2-hydrazino-1-methylpyridine was developed and used in monitoring prostatic 5alpha-dihydrotestosterone, a good index for the follow-up of patients affected by prostate cancer under androgen deprivation therapy and salivary dehydroepiandrosterone, which is now often designated as an anti-aging hormone. A proton-affinitive Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, was used for the determination of 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], a synthetic prodrug of the active form of vitamin D3, in human plasma, and this new LC/positive-APCI-MS method enabled the pharmacokinetic study of 1alpha(OH)D3 in humans. Electron-capture APCI-MS based on derivatization with 2-nitro-4-trifluoromethylphenylhydrazine was used for the analysis of neurosteroids, which affect brain excitability through action at the neurotransmitter receptors. With this method, the stress-induced rapid biosynthesis of pregnane-type neurosteroids in rat brains was demonstrated.

The vitamin D prodrugs 1α(OH)D2, 1α(OH)D3 and BCI-210 suppress PTH secretion by bovine parathyroid cells

Active vitamin D compounds are widely used in the treatment of secondary hyperparathyroidism associated with renal failure. These compounds reduce PTH secretion through vitamin D receptor (VDR)-dependent repression of PTH gene transcription. In previous studies, 1alpha(OH)D3, a vitamin D prodrug, inhibited PTH secretion in cultured bovine parathyroid cells, but it was unclear whether 1alpha(OH)D3 itself or an active metabolite produced this inhibition. We determined the effectiveness of the vitamin D prodrugs 1alpha(OH)D3, 1alpha(OH)D2 and 1alpha(OH)-24(R)-methyl-25-ene-D2 (BCI-210) at inhibiting PTH secretion in bovine parathyroid cell cultures, and examined the metabolism of [3H]1alpha(OH)D2 in these cells. All three prodrugs suppressed PTH secretion with approximately 10% of the activity of 1,25(OH)2D3; much higher activity than expected based on the VDR affinities of these prodrugs (0.25% of 1,25(OH)2D3). Parathyroid cells activated [3H]1alpha(OH)D2 to both 1,25(OH)2D2 and 1,24(OH)2D2. 1,24(OH)2D2 was detectable at 4 h, increased to a maximum at 8 h, and then decreased. In contrast, 1,25(OH)2D2 levels increased linearly with time, suggesting the presence of constitutively active vitamin D-25-hydroxylase not previously reported in parathyroid cells. The cytochrome P-450 inhibitor ketoconazole (50 microM) reduced 1alpha(OH)D2 metabolism to below detectable levels, but did not significantly affect suppression of PTH by 1alpha(OH)D2. The vitamin D prodrugs 1alpha(OH)D3, 1alpha(OH)D2 and BCI-210 suppressed PTH production by cultured parathyroid cells. The ability of 1alpha(OH)D2 to reduce PTH despite inhibition of its metabolism suggests a direct action of this 'prodrug' on the parathyroid gland, but the mechanism underlying this activity is not yet known.

Growth inhibition of carcinogen-transformed MCF-12F breast epithelial cells and hormone-sensitive BT-474 breast cancer cells by 1α-hydroxyvitamin D5

Several studies have established the active form of vitamin D(3) as an effective tumor-suppressing agent; however, its antitumor activity is achieved at doses that are hypercalcemic in vivo. Therefore, less calcemic vitamin D(3) analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), was evaluated for its potential use in breast cancer chemoprevention. Previously, 1alpha(OH)D5 showed anticarcinogenic activity in several in vivo and in vitro models. However, its effects on growth of normal tissue were not known. The present study was conducted to determine the effects of 1alpha(OH)D5 on the growth of normal mouse mammary gland and normal-like human breast epithelial MCF-12F cells and to compare these effects with carcinogen-transformed MCF-12F and breast cancer cells. No significant difference was observed in the growth or morphology of cultured mouse mammary gland and MCF-12F cells in the presence of 1alpha(OH)D5. However, the transformed MCF-12F cells underwent growth inhibition (40-60%, P < 0.05) upon 1alpha(OH)D5 treatment as determined by cell viability assays. Cell cycle analysis showed marked increase (50%) in G-1 phase for cells treated with 1alpha(OH)D5 compared with the controls. Moreover, the percentage of cells in the synthesis (S) phase of cell cycle was decreased by 70% in transformed MCF-12F, BT-474 and MCF-7 cells. The growth arrest was preceded by an increase in expression of cell cycle regulatory proteins p21(Waf-1) and p27(Kip-1). In addition, differential expression studies of parent and transformed MCF-12F cell lines using microarrays showed that prohibitin mRNA was increased 4-fold in the transformed cells. These results indicate that the growth inhibitory effect of 1alpha(OH)D5 was achieved in both carcinogen-transformed MCF-12F and breast cancer cells at a dose that was non-inhibitory in normal-like breast epithelial cells.

Effect of intermittent administration of 200 IU intranasal salmon calcitonin and low doses of 1α(OH) vitamin D3 on bone mineral density of the lumbar spine and hip region and biochemical bone markers in women with postmenopausal osteoporosis: a pilot study

A 1-year prospective, open, randomized, controlled trial was conducted as a pilot study to examine the effect of intermittent administration of 200 IU intranasal salmon calcitonin and 1alpha(OH) vitamin D3 [1alpha(OH)D3] on bone mineral density (BMD) of the lumbar spine and hip as well as on the markers of bone metabolism in women with postmenopausal osteoporosis. A total of 102 randomly recruited women received either 200 IU intranasal salmon calcitonin (Miacalcic nasal 200, Novartis, Basel, Switzerland) daily, 1 month on-1 month off, 0.25 mug 1alpha(OH)D3, and 500 mg elemental calcium continuously (n=57 women) or only 0.25 mug 1alpha(OH)D3 and 500 mg calcium (n=45 women) for a period of 1 year. BMD of the lumbar spine and hip plus biochemical markers reflecting calcium (Ca) metabolism and bone turnover [serum Ca, serum phosphorus, intact parathormone (iPTH), total and bone-specific alkaline phosphatase, osteocalcin levels, 24-h urinary Ca, morning fasting urinary Ca/creatinine, and Pyrilinks-D/creatinine ratio] were measured at the beginning of the study before treatment and after 6 and 12 months of treatment. Baseline characteristics of participants, including age, body mass index, lumbar and hip BMD, and biochemical markers were similar between the two groups. A total of 91 patients completed the study (50 in the salmon calcitonin nasal spray group and 41 in the other group). Lumbar BMD increased significantly in the salmon calcitonin group from baseline (3.0%, p=0.005) and in comparison to the non-calcitonin-treated group (p=0.009). The salmon calcitonin group also had a significant increase in femoral neck BMD compared with baseline values (3.1%, p=0.0005) and in comparison to the non-calcitonin-treated group (p=0.0005) in Ward's triangle BMD (2.9% from baseline values, p=0.009) and in comparison to the non-calcitonin-treated group (p=0.005) in trochanteric BMD (3.4% from baseline values, p=0.007) and in comparison to the non-calcitonin-treated group (P=0.01). Urinary Ca/creatinine and Pyrilinks-D/creatinine levels were significantly decreased from baseline in the salmon calcitonin-treated group (-6.1 and -6.3%, respectively, p=0.001). Bone-specific alkaline phosphatase levels were also significantly decreased from baseline in the salmon calcitonin-treated group (-3.6%, p=0.003). In the same group, a significant decrease in iPTH serum levels compared to baseline values (-2.5%, p=0.005) and in comparison to the non-calcitonin-treated group (p=0.005) was noted. In conclusion, in this pilot study, 1-year intermittent treatment with 200 IU intranasal salmon calcitonin and low doses of 1alpha(OH)D3 produced a significant effect on bone turnover and BMD in postmenopausal women with osteoporosis.

Efficacy and mechanism of action of 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5) in breast cancer prevention and therapy.

It is now well established that the active metabolite of vitamin D3, 1alpha,25(OH)2D3, regulates cell growth and differentiation in various in vitro cancer models. However, its clinical use is precluded due to its hypercalcemic activity in vivo. Hence, several less calcemic vitamin D analogs have been synthesized and evaluated for their chemopreventive and therapeutic efficacy in experimental carcinogenesis models. A novel analog of vitamin D3, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), has currently been under investigation in our laboratory for its application in breast cancer prevention and therapy. 1alpha(OH)D5 had been shown to inhibit development of estrogen- and progesterone-dependent ductal lesions as well as steroid hormone-independent alveolar lesions in a mammary gland organ culture (MMOC) model. Moreover, the inhibitory effect was more significant if 1alpha(OH)D5 was present during the promotional phase of the lesion development. The growth inhibitory effect of 1alpha(OH)D5 has also been manifested in several breast cancer cell lines, including BT-474 and MCF-7. Breast cancer cell lines that responded to 1alpha(OH)D5 treatment were vitamin D receptor positive (VDR+). Vitamin D receptor-negative (VDR-) cell lines, such as MDA-MB-231 and MDA-MB-435, did not show growth inhibition upon incubation with 1alpha(OH)D5. This suggests the requirement of VDR in 1alpha(OH)D5-mediated growth effects. Interestingly, breast cancer cells that were VDR+ as well as estrogen receptor positive (ER+) showed cell cycle arrest and apoptosis, while VDR+ but ER- cells (UISO-BCA-4 breast cancer cells) showed enhanced expression of various differentiation markers with la(OH)D5 treatment. Transcription and expression of estrogen-inducible genes, progesterone receptor (PR) and trefoil factor 1 (pS2), were significantly down-regulated in ER+ BT-474 cells with 1alpha(OH)D5 treatment. This implies a differential effect of 1alpha(OH)D5 on ER+ vs. ER- cells. Additionally, comparison between the effects of 1alpha(OH)D5 on normal vs. transformed cells indicated that 1alpha(OH)D5 does not suppress cell prolifera-

Pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) in normal and uraemic men.

The therapeutic equivalence of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) on the suppression of PTH synthesis and secretion has not clearly been established. The aim of the present study was to evaluate the pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) after oral and i.v. administration in healthy volunteers and uraemic patients. Six healthy volunteers and 12 uraemic patients were included in the study. With an interval of 2 weeks, 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, 4 microg of 1alpha(OH)D(3) i.v. and 4 microg of 1alpha(OH)D(3) orally were administered. Blood samples for analysis of plasma-Ca(2+), plasma-1,25(OH)(2)D(3), and plasma-PTH were drawn at time 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h. The healthy volunteers were studied in all four protocols and the uraemic patients in either the 1alpha(OH)D(3) (n=6) or the 1,25(OH)(2)D(3) (n=6) protocol. After oral administration of 1,25(OH)(2)D(3) the bioavailability of 1,25(OH)(2)D(3) was 70.6+/-5.8/72.2+/-4.8% in healthy volunteers/uraemic patients (n.s.). After i.v. administration the volume of distribution of 1,25(OH)(2)D(3) was similar, 0.49+/-0.14 vs 0.27+/-0.06 l/kg in healthy volunteers vs uraemic patients (n.s.), while the metabolic clearance rate of 1,25(OH)(2)D(3) was 57% lower in the uraemic patients, 23.5+/-4.34 vs 10.1+/-1.35 ml/min in healthy volunteers vs uraemic patients, respectively (P<0.03). The bioavailability of 1,25(OH)(2)D(3) after i.v. administration of 1alpha(OH)D(3) was 42.4+/-11.0/42.0+/-2.0% in healthy volunteers/uraemic patients (n.s.); and after oral administration of 1alpha(OH)D(3) 42.0+/-2.0/29.8+/-3.1% in healthy volunteers/uraemic patients (n.s.). A small, but significant increase in plasma-Ca(2+) was seen after administration of 1,25(OH)(2)D(3) to the uraemic patients, while no increase was seen after administration of 1alpha(OH)D(3). PTH levels were significantly suppressed in the healthy volunteers 24 h after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) orally by 35+/-7, 30+/-8, and 35+/-4%, respectively (all P<0.03). In the uraemic patients, PTH levels were significantly suppressed after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) i.v. by 30+/-10, 45+/-7, and 40+/-7%, respectively (all P<0.04). The effect was transitory in the healthy volunteers and lasted for at least 72 h in the uraemic patients. The present study found a 57% lower metabolic clearance rate of 1,25(OH)(2)D(3) in uraemic patients, as compared with that of healthy volunteers (P<0.03). The bioavailability of 1,25(OH)(2)D(3) following administration of 1alpha(OH)D(3) i.v. and orally in both healthy volunteers and uraemic patients was markedly lower than after administration of oral 1,25(OH)(2)D(3) (P<0.03). In spite of lower plasma-1,25(OH)(2)D(3) levels after administration of 1alpha(OH)D(3), no significant difference was observed on the suppressive effect of 4 microg i.v. of either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) on the plasma-PTH levels in the uraemic patients. This might suggest the existence of an effect of 1alpha(OH)D(3) on the parathyroid glands which is independent of the plasma-1,25(OH)(2)D(3) levels, that are achieved after oral or i.v. administration of 1alpha(OH)D(3).

Effect of alfacalcidol on bone and calcium metabolism in elderly women

Oral administration of active vitamin D3 can reduce the loss of bone mass and the incidence of fractures in patients with osteoporosis in Japan. We conducted a prospective study to confirm the effects of 1 alpha(OH)D3 (Alfacalcidol, Alfarol Chugai Tokyo) on bone and calcium metabolism in elderly women with osteoporosis. Enrolled in the present study were 16 elderly osteoporosis women aged 72.6 +/- 4.5 years to whom 1 microgram of 1 alpha(OH)D3 was administered daily. Fasting blood and urine were obtained at baseline, 1 week, 4 weeks, 12 weeks and 24 weeks after the treatment. Monitored parameters were vitamin D metabolites, intact-PTH, bone alkaline phosphatase (BAP), osteocalcin (OC), deoxypyridinoline (DPD) and pyridiuium crosslinked type I collagen telopeptides (CTx). Serum 1 alpha, 25(OH)2D and PTH levels were significantly increased (p < 0.01) and decreased (p < 0.05), respectively at 1 week after commencing administration. There was a significant decrease of DPD (p < 0.05) at 12 weeks after commencing administration compared to the baseline levels. Serum levels of BAP and OC were found elevated at 1 week, and decreased at 12 weeks. In conclusion, the present study clinically confirmed that 1 alpha(OH)D3 stimulates bone formation in vitro. Long-term administration of 1 alpha(OH)D3 indirectly suppressed bone resorption through the suppression of parathyroid function in the elderly.

Differentiation of human breast carcinoma cells by a novel vitamin D analog: 1alpha-hydroxyvitamin D5.

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3, can induce differentiation in breast cancer cells; however, it is hypercalcemic in vivo. Therefore, development of non-calcemic analogs of vitamin D has received considerable attention. Recently, we synthesized an analog of vitamin D [1alpha(OH)D5] that exhibits much less calcemic activity than 1alpha,25-dihydroxyvitamin D3. In this study, we evaluated the cell-differentiating action of 1alpha(OH)D5 in breast cancer cells. Following 10 days treatment with 1alpha(OH)D5 [(10-7 M) in UISO-BCA-4], we observed induction of intracytoplasmic casein, intracytoplasmic lipid droplets, ICAM-1, nm23, and specific biomarkers associated with breast cell differentiation. 1alpha(OH)D5 treatment also showed induction of vitamin D receptor and TGFbeta1 proteins. UISO-BCA-4 cells pretreated for 10 days in vitro with 1 microM 1alpha(OH)D5 failed to form tumors when transplanted into athymic mice. Similarly, 4 and 8 ng 1alpha(OH)D5 treatment three times weekly inhibited the growth of UISO-BCA-4 cells injected into athymic mice. These results suggest that this new vitamin D analog may be of significant therapeutic value for breast cancer.

1Alpha-hydroxyvitamin D3 prevents the decrease of bone mineral density in lactating beagles.

We assessed the change of bone mineral density (BMD) in lactating beagles with dual energy X-ray absorptiometry (DXA) and the preventive effect of 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) on the BMD. Beagles, two to five years old, were used for detecting the time course change of BMD. Since the coefficient of variation (CV(%)) on detecting lumber vertebral (L2-L4) and tibial BMD by DXA was about 0.5%, DXA was useful to detect the change of BMD in beagles. There was a marked decrease in vertebral BMD during lactational period in the control group. The BMD levels after weaning were found to reverse to the initial level at mating. The same tendency was observed in tibial BMD as vertebral BMD, though the BMD changes were not marked. Beagles were administered at a dose of 0.1 microg/kg of 1alpha(OH)D3 three times in a week, and it was found to suppress the decrease in vertebral BMD during the breast feeding period. Also, the administration of 1alpha(OH)D3 promoted the prevention of decreased BMD during lactation both in vertebrae and tibiae. Significant effects of 1alpha(OH)D3 administration on tibial BMD were not observed. No adverse effects, such as hypercalcemia and hypercalciuria, were observed during the experimental period. Therefore, DXA was useful for detecting the changes of BMD in lactating beagles and the change of BMD was marked in lumber vertebrae, which are rich in trabecular bone. The preventive effect of 1alpha(OH)D3 on the decrease of BMD during the lactation period was observed in beagles.

Inhibition of angiogenesis as a mechanism for inhibition by 1alpha-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 of colon carcinogenesis induced by azoxymethane in Wistar rats.

The effects of 1alpha-hydroxyvitamin D3[1alpha(OH)D3] and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] on the incidence of colon tumors induced by azoxymethane and on the labeling index and angiogenesis of colon tumors were investigated in Wistar rats. Rats received 10 weekly injections of 7.4 mg/kg body weight of azoxymethane and i.p. injections of 1alpha(OH)D3 and 1,25(OH)2D3 at lower and higher doses every other day for 45 weeks. Prolonged administration of both 1alpha(OH)D3 and 1,25(OH)2D3 at a higher dose significantly reduced the incidence of colon tumors in week 45. However, administration of 1alpha(OH)D3 or 1,25(OH)2D3 had little or no effect on the histologic type of colon tumors and cancers. Administration of 1alpha(OH)D3 and 1,25(OH)2D3 at higher doses significantly decreased the labeling index, the immuno-histochemical staining for vascular endothelial growth factor and microvessel counts in colon tumors. Our findings suggest that both 1alpha(OH)D3 and 1,25(OH)2D3 inhibit development of colon tumors. A possible mechanism of inhibition of colon carcinogenesis by 1alpha(OH)D3 and 1,25(OH)2D3 is the inhibition of angiogenesis as well as an anti-proliferative effect.

Secretion of magnesium and calcium in the total saliva ofsheep and its relevance to hypomagnesaemia

The rate of total secretion of saliva was measured over a 3-h period, under steady state conditions, in conscious sheep. The rates of secretion of magnesium and calcium in saliva represented 25-30% of the magnesium and calcium in the extracellular fluid. Since the large stores of intracellular magnesium are not readily mobilizable, the only readily available reserve of magnesium to maintain magnesium homeostasis is a little in bone. Thus, if magnesium ions from food and saliva are not adequately reabsorbed in the fore-stomachs, their loss could contribute significantly to the failure of homeostasis noted in acute ruminant hypomagnesaemia. Studies of the salivary clearance of 28 Mg showed a delay of 5-13 h in the time course of the salivary specific activity of 28 Mg, relative to that in plasma. This delay is indicative of transcellular interchange between magnesium ions in the central compartment, represented by the extracellular fluid, and an outer compartment which includes the salivary epithelial cells. Prolonged hypercalcaemia, caused by injections of 1 alpha (OH) cholecalciferol, was accompanied by a significant increase in the secretion rate of calcium in saliva but the secretion rate of magnesium was unchanged.

25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis.

Renal 25-hydroxyvitamin D3 1alpha-hydroxylase [1alpha(OH)ase] catalyzes metabolic activation of 25-hydroxyvitamin D3 into 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], an active form of vitamin D, and is inhibited by 1alpha,25(OH)2D3. 1alpha(OH)ase, which was cloned from the kidney of mice lacking the vitamin D receptor (VDR-/- mice), is a member of the P450 family of enzymes (P450VD1alpha). Expression of 1alpha(OH)ase was suppressed by 1alpha, 25(OH)2D3 in VDR+/+ and VDR+/- mice but not in VDR-/- mice. These results indicate that the negative feedback regulation of active vitamin D synthesis is mediated by 1alpha(OH)ase through liganded VDR.

Serum bone Gla-protein increases following short-term oral administration of active vitamin D3 in the elderly with vitamin D deficiency

Serum bone Gla-protein (BGP) is produced by osteoblasts in a vitamin K-dependent process. It is well known that serum BGP levels increase after oral administration of active vitamin D3 in postmenopausal women and patients with chronic renal failure before dialysis. These findings indicate that active vitamin D3 increases the BGP production by osteoblasts. In the present study, a daily oral dose of 2 micrograms of 1 alpha (OH)D3 was administered to 10 elderly males (age, 75.0 +/- 10.9 years) and 8 young males (age, 29.0 +/- 3.2 years) to compare the changes in serum BGP levels between the two groups. None of the subjects had severe renal or hepatic dysfunction or abnormalities in calcium (Ca) metabolism. Serum BGP and 1,25(OH)2D levels in both groups were measured before the initial administration, at 24 h and at 1, 2 and 3 weeks after the start of administration. Serum parathyroid hormone (m-PTH), alkaline phosphatase (ALP), Ca and phosphorus levels were also measured in the elderly group. Serum BGP levels before the initial administration were 4.63 +/- 1.8 ng/ml in the elderly group and 4.33 +/- 0.92 ng/ml in the young group, with no significant difference between the two groups. In both group serum 1,25(OH)2D levels were slightly increased at 24 h after administration. In the elderly group serum BGP levels increased significantly to 11.5 +/- 3.0 ng/ml, an increase of approximately 250%, 1 week after the initial administration and the increase was maintained for up to 3 weeks after the administration commenced. However, no changes in serum BGP levels were noted in the young group. In the elderly group serum m-PTH levels were 544 +/- 257 pg/ml, almost the upper normal limit, before, and decreased slightly after, the administration of 1 alpha (OH)D3. Serum ALP levels also decreased slightly after the administration and serum Ca levels were slightly increased, however, the changes were within normal limits. We conclude that osteoblastic function is activated after short-term administration of active vitamin D3 in vitamin D deficient elderly subjects.

The importance of genetic and nutritional factors in responses to vitamin D and its analogs in osteoporotic patients.

The effects of vitamin D and its analogs on fractures and bone mass have been clarified by clinical observations for more than 10 years. Reviewing the results of six clinical trials on osteoporotic fractures using activated vitamin D analogs, there appeared to be a negative correlation between basal levels of calcium intake and the incidence of vertebral fractures in the control groups. For example, when daily calcium intake was about 600 mg, there were approximately 800 vertebral fractures per 1000 persons a year in the controls. When daily calcium intake was above 1000 mg, the incidence was less than 400 fractures per 1000 persons a year. The incidence of fractures decreased by about half in the activated vitamin D-treated group compared with the control group, but the most marked preventive effects of activated vitamin D on fractures were obtained in clinical studies, with daily calcium intakes of 400-800 mg. The effects of vitamin D analogs on bone mass were reported in the clinical studies, but the results are not consistent. However, these studies suggest that the effects of both 1,25(OH)2D3 and 1-alpha(OH)D3 on bone mass were dose dependent, and the doses were low in clinical studies in which good results were not obtained. Significant effects on bone mass were obtained when more than 0.6 micro;g of 1,25(OH)2D3, or more than 0.75 micro;g of 1-alpha(OH)D3 was administered, with increase in the urinary calcium level being within the acceptable range. Reported data indicate that both nonactivated vitamin D and activated vitamin D reduce the serum parathyroid hormone level. However, activated vitamin D administration is more effective, and is able to reduce bone resorption in postmenopausal, osteoporotic patients with a vitamin D-sufficient status. Recent studies concerning the polymorphism of the vitamin D-receptor gene emphasize that sensitivity to active vitamin D varies between genotypes. In the bb type, sensitivity to active vitamin D is high, and calcium absorption efficiency in the intestine under low calcium conditions increases with increase in the serum 1,25(OH)2D level. A significant increase in lumbar bone mineral density was obtained after administration of activated vitamin D to osteoporotic patients of bb type. However, in the genotype with the B factor, sensitivity to active vitamin D was low, and the rate of increase of bone density was low. These data suggest that nutritional and genetic factors are critical when using active vitamin D and its analogs in the treatment of osteoporosis.

Vitamin D analog 25-(OH)-16,23E-Diene-26,27-hexafluoro-vitamin D3 induces differentiation of HL60 cells with minimal effects on cellular calcium homeostasis.

Numerous vitamin D3 analogs (VDAs) can inhibit the proliferation of cells from several types of human malignancies. The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3(1,25D3), is formed by successive hydroxylations of cholecalciferol at the 25 and 1 alpha positions. In this study we examined the effects of the absence of the 1 alpha (OH) group, introduction of a double bond in position 16, and further modifications at the 23, 26, and 27 positions in the side chain on the potency of the VDAs. The parameters studied were the rapidity of the induction of monocytic differentiation, the cell cycle traverse, and the effects of VDAs on intracellular calcium homeostasis in HL60 cells. The results show that (1) 1,25D3 derivatives which lace the 1 alpha (OH) group have little differentiation-inducing activity, (2) hexafluorination (6F) of the terminal methyl groups in the side chain partially restores the activity of 1 alpha-desoxy compounds and potentiates the activity of 1 alpha hydroxylated compounds, and (3) 25-(OH)-16,23E-diene-26,27-hexafluoro-vitamin D2 (Ro25-9887) alone among the twelve compounds tested induces differentiation with only minimal changes in the basal levels of intracellular calcium and store-dependent calcium influx in HL60 cells. Addition of 1 alpha (OH) group to this compound increases its differentiation-inducing activity but also elevates basal calcium level. The results suggest that altered calcium homeostasis is not an obligatory component of HL60 leukemia cell differentiation, and that Ro25-9887 and related VDAs may be suitable for testing as components of anti-leukemic therapy.