EHDP-treated Rats Research Articles

Vitamin D3 increased intestinal Na/Pi-IIb and CYP27B1 mRNA level in rats fed low-phosphorus diets.

The objective of the study was to determine the role of vitamin D3 (VD3) in regulating adaptation and mechanism of rats to low-phosphorus (P) diets. Rats were assigned to 4 diets containing 0.2%, 0.4%, 0.6%, or 0.8% P consisting of 5 replicate cages with 6 rats per replicate cage and fed for 7 days. Four rats from each replicate cage were treated with ethane-1-hydroxy-1,1-diphosphonicacid, tetrasodium salt (EHDP) and 2 rats remained untreated. Twelve hours prior to preparation on d 7, two of the EHDP-treated rats received an intraperitoneal injection of VD3 [1,25-(OH)2D3] at 600 ng per kg body weight, while two rats did not receive the injection. Rats that did not receive VD3 injection had decreased (P < 0.001) P absorption, but injection of VD3 resulted in increased (P < 0.001) absorption. The effect of VD3 injection was greater (P < 0.001) for rats fed 0.2% P diet than rats fed 0.8% P diet in ileum. Sodium dependent phosphate cotransporter type Ⅱb (Na/Pi-II b) and 25-hydroxyvitamin D 1-α hydroxylase (CYP27B1) mRNA level showed the same trend with P absorption. Serum concentration of VD3 and 1α-hydroxylase activity in rats fed 0.2% P diet were lower than those fed 0.8% P diet. The injection of VD3 increased (P < 0.001) serum concentration of VD3 and 1α-hydroxylase activity. Thus, VD3 increased Na/Pi–IIb and CYP27B1 mRNA level and improved serum concentration of VD3 and 1α-hydroxylase activity in rats fed low-P diets.

EHDP‐induced rachitic syndrome in rats is not reversed by vitamin D metabolites

To examine whether either of the two known active vitamin D metabolites 1,25(OH)2D3 or 24,25(OH)2D3 could reverse the mineralization defect induced by 1-hydroxyethylidene-1,1-bis phosphonate (EHDP), a model of EHDP-induced rickets was used. Rats at the age of 31 days were injected for 10 consecutive days with EHDP (10 mg/kg). Other littermates were treated with a combination of EHDP and either 1,25(OH)2D3 or 24,25(OH)2D3 or were treated following 10 days of EHDP, with either of the vitamin D metabolites for an additional 72 hr. Samples of cartilage fluid (Cfl) and of blood were removed prior to sacrifice for biochemical studies of some parameters of calcification. These parameters were correlated with the results of light and electron microscope studies of growth plate cartilage and bone. EHDP-treated rats revealed signs of typical rickets, manifested by widened growth plates and impaired bone mineralization. Transmission electron microscope (TEM) examination revealed matrix vesicles distributed throughout the growth plate; however, there appeared to be an arrest of the spread of the crystals at the provisional zone of calcification. Treatment with either 1,25(OH)2D3 or 24,25(OH)2D3 failed to reverse the rachitic condition of the animals. Serum calcium blood levels were elevated in the 1,25(OH)2D3 and EHDP-treated group. 1,25(OH)2D3 and 24,25(OH)2/D3 further increased the already elevated serum alkaline phosphatase levels observed in EHDP rats, although the increase observed with 1,25(OH)2D3 was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma levels of vitamin D metabolites in diphosphonate-treated rats.

1. Protein-binding assays have been used to measure plasma 1,25-dihydroxy-vitamin D [1,25-(OH)2D] as well as 25-hydroxy-vitamin D [25-(OH)D] in rats given 10 mg of phosphorus (P) day(-1) kg(-1) as ethane-1-hydroxy-1,1-diphosphonate (EHDP). 2. In control animals given a normal laboratory chow plasma 25-(OH)D and 1,25-(OH)2D were about 40 nmol/l and 300 pmol/l respectively. 3. EHDP produced a decrease of plasma 1,25-(OH)2D to below 50 pmol/l in 2 days. 4. Both in control and in EHDP-treated rats plasma 1,25-(OH)2D increased when dietary calcium (Ca) was restricted to 0.1%, or dietary P to 0.2%, indicating that the well-known stimulatory effect of Ca or P deprivation was at least partially effective in EHDP-treated rats. 5. In response to an increase of the oral supply of vitamin D3 to 65 nmol/day the plasma level of 25-(OH)D rose in both control and EHDP groups. Plasma 1,25-(OH)2D was not increased above the normal value in control rats. In EHDP-treated rats, however, plasma 1,25-(OH)2D rose to a level equal to that in controls, suggesting that the effect of EHDP on plasma 1,25-(OH)2D can be overcome at high precursor concentration.

Inhibition of diphosphonate-blocked bone mineralization. Evidence that calcitonin promotes mineralization.

There is much experimental evidence which indicates that calcitonin in hibits bone mineral resorption, but there are few data available in support of the proposal that calcitonin may also promote mineralization. Ethane-1-hydroxy-1,1-diphosphonate (EHDP) administered to immature rats inhibited mineralization as evidenced by widened tibial epiphyseal plates and decreased bone ash to dry weight ratios. Concurrent dosing with salmon calcitonin (SCT) prevented or reversed the EHDP-blocked mineralization in a dose dependent manner. Administration of SCT during the period after EHDP treatment significantly improved mineralization of tibial epiphyseal plates as shown by plate width narrowing and increased uptake of radioactive calcium. These results suggest that SCT increased mineralization in EHDP-treated rats, and provide supportive evidence for the proposal that calcitonin may also promote mineralization, in addition to its well known ability to inhibit bone mineral resorption.

Evidence for the Promotion of Bone Mineralization by 1α,25-Dihydroxycholecalciferol in the Rat Unrelated to the Correction of Deficiencies in Serum Calcium and Phosphorus

Concurrent administration of 1alpha,25-dihydroxycholecalciferol [1alpha,25-(OH)2-CC] to intact and thyroparathyroidectomized rats treated with ethane-1-hydroxy-1,1-diphosphonate (EHDP) prevented or reversed the EHDP-induced inhibition of bone mineralization as measured by changes in epiphyseal plate width and ash content of bone. An analog, 1alpha-droxycholecalciferol, was also effective. Recovery of bone after EHDP treatment was also significantly improved by administration of 1alpha,25-(OH)2-CC as evidenced by enhanced uptake of 45Ca by epiphyseal plates and decreased plate widths. Cholecalciferol (CC), ergocalciferol, dihydrotachysterol2, 5,6-trans-CC, 25-OH-CC, 5,6-Trans-25-OH-CC, and 1alpha24R,25-(OH)3-CC also blocked EHDP-induced epiphyseal plate widening, but required high, pharmacological dose levels. 24R,25- (OH)2-CC was inactive at doses up to 10 microgram/day. Since EHDP-treated rats are not deficient in calcium or phosphate, these data suggest that 1alpha,25-dihydroxycholecalciferol promoted bone mineralization independently of effects upon the intestinal absorption of calcium and phosphate.

Distribution of 99Tcm-labelled phosphorus compounds, 45Ca and 85Sr in diphosphonate-treated rats.

A similar distribution of 99Tcm, 45Ca and 85Sr was obtained when injected in rats as 99Tcm-labelled pyrophosphate or ethylene-1-hydroxy-1,1-diphosphonate (EHDP), 45Ca-chloride and 85Sr-choloride respectively. The highest concentrations occurred in bone and teeth. In EHDP-treated rats, isotopes were accumulated at sites of ectopic calcifications in the lung, kidney and stomach wall as well as in mineralized tissues. These results indicate that 99Tcm, when administered as 99Tcm-labelled EHDP accumulates predominatly in the inorganic rather than organic tissue components.

Ethane-1-hydroxy-1, 1-diphosphonate (EHDP). Effects on growth and modeling of the rat tibia.

One hundred gram male Sprague-Dawley rats were divided into groups which were injected daily for 10 or 30 days with vehicle (control group), 0.2, 0.4, 2.0, or 10.0 mg ethane-1-hydroxy-1,1-diphosphonate (EHDP)/kg/day. The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation factors. Knowing these, information about hard tissue resorption was deduced. After ten or thirty days treatment with 2.0 or 10.0 mg EHDP/kg/day, there was an increase in percentage of hard tissue. This was due to a decrease in bone formation with a greater decrease in hard tissue resorption. Furthermore, after thirty days treatment with 0.2 and 0.4 mg EHDP/kg/day, there was an increase in percentage of hard tissue, which was due to a decrease in resorption with no change in formation. Ultrastructural studies on osteoclasts from EHDP-treated rats showed a general decrease in their vacuolization and amount of organelles as dosage of EHDP increased. Histologic findings suggest that EHDP is similar to fluoride in the way in which it depresses hard tissue resorption.

Separation of the hypocalcemic and hypophosphatemic actions of calcitonin with disodium ethane-1-hydroxy-1,1-diphosphonate.

The present studies were designed t o determine the effects of ethane-1,1-diphos-phonic acid (EHDPTM), an analog of pyrophos-phate, on the hypocalcemic and hypophosphatemic actions of salmon calcitonin (SCT). Rats were pretreated for 3 or 4 days with daily subcutaneous (sc) injections of EHDP (40 mg/kg body wt). 45Ca was administered (intraperitoneally) 10 days prior to EHDP treatment and 32P (intravenously) the day following the last injection of the drug. SCT (0.4 MRC mU/g body wt) was injected sc 1 hr after 32-P administration. Preliminary tests with thfS drug had confirmed that the dosage used at least partially inhibited the 45Ca removal from bone stimulated by low-calcium diet or parathyroid hormone administration; and at least partially reduced the uptake of both 45Ca and 32P by bone, during the first 5 hr after intravenous administration of isotopes. Following SCT injection plasma calcium concentrations fell only 5% in EHDP-treated rats compared to 25% in rats not treated with EHDP. Plasma phospha...

Studies on dentinogenesis in the rat

The effects of ethane-1-hydroxy-1,1-diphosphonate (EHDP) in vivo on crystal growth in the early stages of predentin calcification were studied in newborn Sprague-Dawley rats after intraperitoneal injection of 0.1 ml physiological saline solution containing EHDP at a concentration of 100 or 500 µg/ml. The animals were given one injection per day for four consecutive days, thus receiving a total amount of 8 or 40 mg EHDP respectively. Control rats were injected with the same volume of physiologic saline alone. The following electron microscope observations were made: In dentin, the EHDP-treated rats were found to exhibit crystallites of smaller size than the normal controls. In the mineralization zone, most of the needle-like crystallites did not reach 20 A in width. Their further growth was inhibited, only a few reaching 25 A in width in the intertubular dentin. Within extracellular membrane-bound bodies or “dentinal globules” normally found in early formed predentin matrix, needle-like crystallites appeared before mineral deposits were visible in the predentin matrix. The crystallites had a paired appearance with a central electron-lucid core. The crystallite pattern was disturbed in EHDP-treated rats. Although less pronounced, similar changes were found also in rats given the lower dose of EHDP.

Studies on dentinogenesis in the rat. Ethane-1-hydroxy-1,1-diphosphonate (EHDP) inhibits crystal growth in predentin calcification.

The effects of ethane-1-hydroxy-1,1-diphosphonate (EHDP) in vivo on crystal growth in the early stages of predentin calcification were studied in newborn Sprague-Dawley rats after intraperitoneal injection of 0.1 ml physiological saline solution containing EHDP at a concentration of 100 or 500 µg/ml. The animals were given one injection per day for four consecutive days, thus receiving a total amount of 8 or 40 mg EHDP respectively. Control rats were injected with the same volume of physiologic saline alone. The following electron microscope observations were made: In dentin, the EHDP-treated rats were found to exhibit crystallites of smaller size than the normal controls. In the mineralization zone, most of the needle-like crystallites did not reach 20 A in width. Their further growth was inhibited, only a few reaching 25 A in width in the intertubular dentin. Within extracellular membrane-bound bodies or “dentinal globules” normally found in early formed predentin matrix, needle-like crystallites appeared before mineral deposits were visible in the predentin matrix. The crystallites had a paired appearance with a central electron-lucid core. The crystallite pattern was disturbed in EHDP-treated rats. Although less pronounced, similar changes were found also in rats given the lower dose of EHDP.

Influence of disodium ethane-1-hydroxy-1,1-diphosphonate on vitamin D metabolism in rats.

The metabolism and the organ distribution of double labelled vitamin D3 (1,2-3H-4-14C-cholecalciferol) has been studied in rats in which the bone mineralization and the intestinal calcium absorption have been inhibited by a large pose (10 mg P/kg s.c. for 7–14 days) of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The most striking difference found was a reduced accumulation of radioactive cholecalciferol and its metabolites in the kidney of EHDP-treated rats. It is unlikely that this effect was due to an unspecific alteration of the functional renal tissue since blood urea, glomerular filtration rate and renal plasm a flow remained unaltered by this dose of EHDP. The EHDP-treated rats were able to form the metabolite eluted with peak IV of the silicic acid chromatographic system, that is 25-hydroxycholecalciferol. In these vitamin D repleted rats fed a high calcium diet, the tritium deficient metabolite eluted with peak V (1,25-dihydroxycholecalciferol) was only found in the intestinal mucosa of both control and EHDP groups three days after the injection of radioactive cholecalciferol, and this in a very small amount. Therefore no definitive conclusion can be drawn as to a possible interference of EHDP treatment on the production of 1,25-dihydroxycholecalciferol. The change in the renal metabolism of vitamin D in rats treated with a rachitogenic dose of EHDP may be caused by the modifications of the calcium metabolism brought about by the diphosphonate. Its relation, if any, with the decreased calcium absorption remains to be established.