Effect of boron on vitamin D deficient rats

The effects of different levels of dietary boron were determined in vitamin D deficient rats. Vitamin D deficient diets containing either 0.158 ppm or 2.72 ppm of boron were fed to rats for 11 w, and calcium, magnesium, and phosphorus apparent absorption and balance were measured in the twelfth week. Higher apparent absorption and balance values for calcium and phosphorus were observed in the rats with higher dietary boron, but very few differences were seen in body wt, organ wt, and bone parameters. Balance measurements represented the present status of the rats after 12 w on the diets, but other measurements represented an accumulation over the lifetime of the rat, including a suckling period with ample vitamin D and boron. The data demonstrated that when rats are vitamin D deficient, as indicated by hypocalcemia, the level of boron in the diet affects mineral balance.     

Calcium uptake into renal brush border membranes in vitamin B6 deficient rats

The calcium uptake into renal brush border membrane vesicles, which has been purified from normal or vitamin B6 deficient rat renal cortex by calcium precipitation, was investigated. The values of Km and Vmax were determined to be 1.89 mM and 4.26 nmol of Ca2+/mg of protein per 20s in vitamin B6 deficient rats, respectively. This Vmax was lower than that of normal rats. The chemical compositions of renal brush border membranes did not display a difference in normal and vitamin B6 deficient rats. The amount of brush border membranes isolated from 1 gram of renal cortex in vitamin B6 deficient rats was less than in normal rats.     

Protein secretion from parotid glands of vitamin D deficient and glucocorticoid-treated rats

The rate of isoproterenol stimulated secretion of protein from parotid glands of vitamin D deficient rats and rats treated with methylprednisolone was increased compared to the secretory response of tissue from control rats. It is suggested that the increased secretory response is secondary to a decreased capacity of mitochondria from the tissue of these animals to take up and store Ca²⁺; i.e. the mitochondria are less efficient buffers of cytoplasmic Ca²⁺. Under these conditions any process, such as protein secretion, which requires an increased cytoplasmic Ca²⁺ concentration will operate more effectively.     

Changes of femoral alkaline phosphatase activity in adult rats treated by sorbitol enriched or vitamin D3 deficient diet

The effect of vitamin D3-deficiency and dietary sorbitol on serum calcium level, the activity and alkaline phosphatase (AP) pattern in femoral epiphysis were studied. Rats fed a diet supplemented with sorbitol or vitamin D3 showed the same serum calcium concentration and AP activity in serum and femur. Rats fed a vitamin D3-deficient diet displayed decreased serum calcium concentration and increased AP activity both in serum and femur. Four forms of AP were isolated from the femur of these rat groups: of Mr 100,000, 110,000, 130,000 and 165,000. Rats receiving the diet supplemented with sorbitol showed a marked rise in the activity of the Mr 165,000 form, and appearance of a new monomer of 100,000, never formed in two remaining groups.     

An estimation of the parathyroid hormone secretion rate in vitamin D deficient rats.

In an attempt to define the rate of endogenous secretion of parathyroid hormone in vitamin D deficiency, graded amounts of hormone were infused constantly into vitamin D deficient and vitamin D fed acutely thyroparathyroidectomized rats in a perfusion balance study. The dose of hormone needed to compensate for acute thyroparathyroidectomy was estimated. Serum levels of calcium and phosphorus, renal clearances of phosphorus and urinary excretion of cyclic AMP, each determined 16 hr after the surgery, were employed as parameters of the hormone effect. It was concluded that, in the condition of the perfusion balance study, the secretion rate of parathyroid hormone was 5.1-8.6 USP u/hr in the vitamin D deficient animals compared with 0.81-0.84 USP u/hr in the control.     

Regulation of the vitamin D receptor and cornifin beta expression in vaginal epithelium of the rats through vitamin D3

The aim of the present study was to determine the respective role of 1,25-dihydroxyvitamin D3 on vaginal epithelium and 1,25-dihydroxyvitamin D3 receptor expression in ovariectomized rats and vitamin D3 treated rats. Bilateral ovariectomies were performed in 20 mature, non-pregnant Wistar female rats. All the animals were divided into 2 groups consisting of 10 rats each. Group I served as control. In group II, animals were injected intramuscularly with vitamin D3 (50, 00 IU/kg). Two weeks after the injections, vaginas of animals in group I and group II were removed removed and processed for immunohistochemistry. Epithelial differentiation, 1,25-dihydroxyvitamin D3 receptor and cornifin beta expression were investigated in vaginal epithelium of control group (ovariectomized) and vitamin D3 treated rats. Vaginal epithelial cells from vitamin D3 treated animals changed into highly- stratified keratinizing layers. 1,25-dihydroxyvitamin D3 receptor and cornifin beta as a marker of squamous differentiation were present in ovariectomized rats treated with 1,25-dihydroxyvitamin D3. In contrast, cornifin beta and 1,25-dihydroxyvitamin D3 receptor were absent in all layers of vaginal epithelium in control group. We demonstrated for the first time that 1,25-dihydroxyvitamin D3 induced proliferation of vaginal epithelium consistent with the cornifin beta expression and 1,25-dihydroxyvitamin D3 up-regulated 1,25-dihydroxyvitamin D3 receptor expression in vaginal epithelium.     

Epigenetic regulation of vitamin D hydroxylase expression and activity in normal and malignant human prostate cells

It was previously suggested that the 25-Vitamin-D₃-1-hydroxylase (CYP27B1) is downregulated during human prostate tumor pathogenesis while the catabolic 25-Vitamin-D₃-24-hydroxylase (CYP24) expression is increased. The latter could lead to resistance against the antimitotic, prodifferentiating activity of 1,25-dihydroxycholecalciferol. Our hypothesis was that regulation of Vitamin D hydroxylase expression during prostate tumor progression might be under epigenetic control. We demonstrate by real time RT-PCR that PNT-2 human normal prostate cells indeed possess CYP27B1, but are practically devoid of CYP24 mRNA, whereas DU-145 cancer cells have constitutive expression of CYP24, and very low levels of CYP27B1 mRNA. Treatment of PNT-2 cells with the methylation inhibitor 5-aza-2′-deoxycytidine together with the deacetylation inhibitor trichostatin A resulted in elevation of both CYP27B1 and CYP24 mRNA expression demonstrating that even in normal human prostate cells expression of Vitamin D hydroxylases may be under epigenetic control. In the DU-145 malignant cell line trichostatin A together with 5-aza-2′-deoxycytidine increased CYP27B1 mRNA expression to a smaller extent than in normal cells, however this resulted in a highly significant increase in 1-hydroxylation capacity. This demonstrates for the first time that synthesis of 1,25-dihydroxycholecalciferol in human prostate tumors could be reinitiated by epigenetic regulators.     

Increased plasma lipidperoxidation in vitamin B-6 deficient rats

Lipidperoxidation in plasma of rats fed with vitamin B-6 deficient diet for a period of 12 weeks was studied with pair-fed controls. Plasma pyridoxal 5'-phosphate, alanine amino transferase and aspartate amino transferase, the markers of vitamin B-6 status, were significantly low in vitamin B-6 deficient rats. Plasma malondialdehyde level, conjugated dienes and lipofuscin like pigments were increased in vitamin B-6 deficiency. Increased levels of plasma lipids, calcium, iron and copper were observed in vitamin B-6 deficiency. Plasma susceptibility to lipidperoxidation was maximal in vitamin B-6 deficiency, upon stimulation by the promotors, Fe2+, Fe3+, Cu2+, ascorbate, t-butyl hydroperoxide and hydrogen peroxide.     

Regulation of vitamin D metabolism.

Vitamin D can be regarded as a prohormone and its most potent metabolite, 1, 25-dihydroxyvitamin D₃, a hormone which mobilizes calcium and phosphate from bone and intestine. In true hormonal fashion, the biosynthesis of 1, 25-dihydroxyvitamin D₃ by kidney mitochondria is feed-back regulated by serum calcium and serum phosphorus levels. The lack of calcium brings about a secretion of parathyroid hormone which stimulates 1, 25-dihydroxyvitamin D₃ synthesis while low blood phosphorus stimulates 1, 25-dihydroxyvitamin D₃ synthesis even in the absence of the parathyroid glands. For such regulation to occur, vitamin D must be present probably because 1, 25-dihydroxyvitamin D₃ itself is needed for the regulation. The molecular and cellular mechanisms whereby 1, 25-dihydroxyvitamin D₃ synthesis is regulated are unknown despite many recent reports. Likely the elucidation of these mechanisms must await a detailed investigation of the enzymology of the renal 25-hydroxyvitamin D₃-1α-hydroxylase. In addition to the regulation at the 25-hydroxyvitamin D₃-1α-hydroxylase step, vitamin D metabolism is regulated at the hepatic vitamin D-25-hydroxylase level. This regulation is a suppression of the hydroxylase by the hepatic level of 25-hydroxyvitamin D₃ itself by an unknown mechanism. Much remains to be learned concerning the regulation of this newly discovered endocrine system but already the findings are not only relevant to calcium homeostasis but also to an understanding of a variety of metabolic bone diseases.     

Changes in pineal indoleamine metabolism in vitamin A deficient rats

Weanling, male rats were fed a vitamin A deficient (VAD) diet from 20 to 77 days of age. The circadian rhythms of the precursors and metabolites of pineal melatonin were measured along with the activity of N-acetyltransferase (NAT). Significant decreases in peak melatonin levels (0100 hours) and in nightime NAT activity (0100 and 0300 hours) were found in the pineals of the VAD rats. In contrast, the contents of serotonin, 5-hydroxytryptophan and 5-hydroxyindole acetic acid were only moderately affected by the deficiency. Daily administration of 25 micrograms melatonin from 20 to 74 days of age markedly reduced NAT activity in control and VAD rats. These data suggest that NAT activity is more sensitive to chronic VAD than any other parameters of melatonin metabolism.     

Regulation of vitamin D metabolism by calcium and phosphate ions in isolated renal tubules.

The acute and long-term effects of Ca2+ and Pi on vitamin D metabolism were studied in vitro with isolated renal tubules from vitamin D-deficient and vitamin D-supplemented chicks. Ca2+ depletion, achieved by isolating renal tubules in Ca2+-free buffers, led to suppression of 1 alpha-hydroxylase activity. Re-introduction of Ca2+ during incubation caused an acute stimulation of this enzyme. This stimulatory effect of Ca2+ was prevented by prior treatment of Ca2+-depleted renal tubules for 6 h with 1,25-dihydroxycholecalciferol. Ca2+ and Pi produced marked acute affects on 1 alpha-hydroxylase activity, which persisted for the whole 8 h experimental period, in Ca2+-depleted renal tubules from vitamin D-deficient chicks. The effects of either ion were influenced by the concentration of the other. However, the effects of these ions could not be reproduced in either Ca2+-depleted renal tubules from vitamin D-supplemented chicks or in renal tubules from vitamin D-deficient chicks, isolated in Ca2+-containing buffers. Isolation of renal tubules from vitamin D-supplemented chicks in Ca2+-containing buffers and subsequent incubation for 8 h in the presence of increased [Ca2+] led to a modest but statistically significant suppression of 1 alpha-hydroxylase and stimulation of 24-hydroxylase activity. It is concluded that the acute effects of Ca2+ and Pi on 1 alpha-hydroxylase activity of Ca2+-depleted renal tubules from vitamin D-deficient chicks are not regulatory but the results of the experimental conditions. In contrast the long-term effects of Ca2+ on both hydroxylases of renal tubules from vitamin D-supplemented chicks may be of physiological significance.     

Effect of ipriflavone on bone changes induced by calcium restricted, vitamin D deficient diet in rats

The preventive effect of ipriflavone, 7-isopropoxy-isoflavone, on the development of experimental osteopenia in rats was studied. Male Wistar rats (4 weeks old) on a calcium restricted, vitamin D deficient diet were given a daily oral administration of ipriflavone. The administration of ipriflavone (100 mg/kg BW/day) for 40 days significantly inhibited a decrease in the cortical thickness (14.0 +/- 1.6 vs. 17.1 +/- 2.9%, mean +/- SD, p less than 0.05) and bone calcium content (62 +/- 4 vs. 67 +/- 2 mg, p less than 0.05) in the femora of rats induced by a mild calcium restricted (0.3%), vitamin D deficient diet. This compound did not affect serum calcium levels in this condition. But a dose of 20 mg/kg BW/day of ipriflavone was insufficient to inhibit a decrease in bone calcium content. In rats fed on a more severe calcium restricted (0.03%), vitamin D deficient diet, the administration of ipriflavone (100 mg/kg BW/day) did not significantly affect the cortical thickness or calcium content. Intestinal calcium absorption measured by the in situ loop method was not significantly different between rats fed with a severe calcium restricted (0.03%), D deficient diet with or without ipriflavone (20 or 100 mg/kg BW/day) These results demonstrate that the new compound, ipriflavone, partially prevents bone calcium loss induced by a mild calcium restricted (0.3%), vitamin D deficient diet in rats. However, the precise mechanism of action of this compound remains unknown.     

Radiopharmacological profiles of tracers under pathophysiological conditions. Bone uptake of 99mTc-phytate in vitamin D deficient rats

The radiopharmaceutical ^99mTc-phytate, formed by reacting the phosphatic ligand, myo-inositol hexakisphosphate with a reduced form of ^99mTcO₄⁻, is widely employed in diagnostic nuclear medicine for scinti-imaging the RE system. Despite it being a compound derived from a ligand containing phosphatic functional moieties it does not concentrate to a significant extent in the osseous tissue following i.v. injection into animals or humans. Neither has there been any clinical report citing its localization in the skeletal tissue consequent to its i.v. injection into patients with different types of pathophysiological conditions/metabolic bone disorders. In the course of our study of the homing characteristics of radiopharmaceuticals into specific organ systems or secondary target sites we found that ^99mTc-phytate could be directed to some extent into the skeletal tissues of experimental rats by altering its physiological milieu—viz. by decreasing blood calcium levels and increasing bone resorption. Such an altered physiologic condition is obtained by experimentally inducing vitamin D deficiency in animals. Our results show that 8–10% of the administered dose could be redirected to the bone matrix but at the expense of the liver. The study also indicates that many so-called organ specific ^99mTc-radiopharmaceuticals could have more than one target tissue/organ system for accumulation consequent to being introduced into the systemic circulation. In a number of instances the secondary target may be masked and may be elicited only under certain specific pathophysiologic conditions.