Local delivery of an amino bisphosphonate prevents the resorptive phase of alveolar bone following mucoperiosteal flap surgery in rats.

Mucoperiosteal flaps are used to access the bone and root surface in a wide range of periodontal procedures and in implant surgery. We have demonstrated that the mucoperiosteal surgical flap of the rat mandible produces a transient burst of alveolar bone resorption similar to the clinical observations in humans. This resorptive activity, when coupled with local irritation factors, may cause confined alveolar bone loss. Recently, we have demonstrated that an amino bisphosphonate, which is used in preventing systemic bone resorption in osteoporosis and other bone diseases, reduces alveolar bone resorption in the rat model when administered systemically. In this study we evaluated the effect of local delivery of the amino bisphosphonate on bone resorption associated with mucoperiosteal flaps. Following mucoperiosteal flap elevation in the premolar and molar region of the rat mandible, a surgical pellet soaked with amino bisphosphonate was locally applied on the exposed bone surface and covered by flap. The results show that local delivery of amino bisphosphonate reduces significantly alveolar bone resorption activated by mucoperiosteal flap surgery. This study suggests that local application of amino bisphosphonate can be used as an adjunct in therapy for reducing bone resorption following surgery.

Acute stimulation of creatine kinase activity by vitamin D metabolites in the developing cerebellum.

There is increasing evidence that vitamin D metabolites have a developmental function. We have investigated the influence of the vitamin D status on the activity of creatine kinase in the brain. Normally fed rats show an increase in the specific activity of cerebral and cerebellar creatine kinase during postnatal development. Vitamin-D-depleted rats failed to show this normal increase. Developing cerebellum, but not cerebrum, in both vitamin D-depleted rats and in normally fed animals, responded sequentially to a single injection of a vitamin D metabolite by displaying increased creatine kinase specific activity. In 5-25-day-old rats, 24R,25-dihydroxyvitamin D-3 significantly increased creatine kinase specific activity 24 h after injection. In contrast, 1,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity from 20 days after birth. A similar pattern of sequential responsiveness to vitamin D metabolites, but at an earlier age, was shown in the cerebellum of the rabbit, which is a 'perinatal brain developer' compared to the rat, a 'postnatal brain developer'. Because of the difficulty in obtaining vitamin D-depleted rabbits, studies were carried out in normally fed animals. In these rabbits, 24R,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity between 6 days before birth and 9 days after birth, while 1,25-dihydroxyvitamin D-3 caused an increase in cerebellar creatine kinase specific activity from 8 days after birth. These developmental differences found in creatine kinase basal activity and responsiveness are correlated with differences in cellular growth rates, both in the rabbit and in the rat, suggesting that vitamin D metabolites may be required for optimal cerebellar development.

Vascular placental insufficiency in the rabbit. Changes in creatine kinase and adenylate kinase activities in fetal tissues.

Vascular placental insufficiency is considered a common pathogenic factor in human intrauterine growth retardation. To mimic this condition, the rabbit, a 'perinatal brain developer' was utilized as an experimental model. Ischemic conditions were achieved by total ligation of approximately 30% of the uteroplacental vessels of half of the fetuses in each pregnant rabbit in the last third of gestation. The change in activity of the brain type isozyme of creatine kinase (CKBB), involved in energy regeneration and regulation, was assessed as a response marker to tissue ischemia in rabbit tissues: cerebellum, cerebrum, kidney, liver and placenta. A significant transient increase in CK-specific activity was found in the kidney and the cerebellum but not in the other organs tested, at 24 and 48 h after ligation. This increase was not seen with adenylate kinase, another enzyme involved in energy regeneration and regulation. It is proposed that an increase in CK-specific activity could serve as a metabolic marker of vascular insufficiency in rapidly developing tissues, representing part of a compensatory mechanism to overcome an energetic gap induced by ischemia.

Pharmacokinetic changes of several antibiotics in chickens during induced fatty liver.

The concentrations of five antibiotics (erythromycin, lincomycin, penicillin G, streptomycin and oxytetracycline) were determined in chicken serum before and after induced fatty liver. The pharmacokinetic variables were calculated according to the obtained data. The crossover trial design involved 10 chickens for each antibiotic. The fatty liver was produced by oestradiol-dipropionate injections and monitored by serum malic enzyme activity determinations. Protein binding of the respective antibiotics was determined in vitro in the serum obtained from normal and oestrogen-treated birds. Induction of fatty liver caused several changes in the determined variables. The measured peak concentrations were higher for lincomycin and erythromycin and lower for penicillin and oxytetracycline while streptomycin remained unchanged. The peak concentration of streptomycin appeared earlier and the peak of oxytetracycline later than in the normal chickens. The elimination half-lives were shorter for erythromycin, lincomycin and streptomycin and increased for penicillin and oxytetracycline. The area under the concentration curve (AUC) decreased for erythromycin, penicillin and streptomycin, increased for oxytetracycline and remained unchanged for lincomycin. The body clearance (ClB/f) and the apparent specific volume of distribution (Vd(area'/f) were considerably changed in association with fatty liver induction. Since the fraction of the drug absorbed (f) is not known, it can only be speculated that changes in distribution rather than reduced liver function altered the kinetics. The protein binding was decreased for all the antibiotics, but this did not seem to be the reason for changes in kinetics, except perhaps in the case of penicillin.

Developmental changes in responsiveness to vitamin D metabolites.

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

Effect of parathyroid hormone and uremia on erythrocyte deformability.

Parathyroid hormone (PTH) caused a significant decrease in human erythrocyte filtration rate (EFR). This effect was Ca2+-dependent and was partially reversed by the Ca2+ blocker verapamil. It was mimicked by the Ca2+ ionophore A-23187. Mg2+ even at high concentrations could not substitute for Ca2+. There was a dose response between the filtration rate and Ca2+ or PTH concentrations. Serum ultrafiltrate of patients with chronic renal failure and secondary hyperparathyroidism caused significant inhibition similar to that seen with PTH extract. Ultrafiltrate from patients with chronic renal failure following parathyroidectomy and from healthy individuals did not cause this phenomenon. Erythroyctes deformability in uremic patients appears to be caused by high PTH levels. Our present findings agree with ones about the toxic effects of PTH on various biological systems.

Developmental changes in the responsiveness of rat kidney to vitamin D metabolites.

Kidneys from both normal and vitamin D-deficient rats were found to show changes in responsiveness to vitamin D metabolites during postnatal development, correlated with the concentrations of the specific receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the specific binding protein for 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Cytosol preparations from kidneys of vitamin D-deficient rats, in the second week of life, contained specific binding proteins for 24,25-(OH)2D3. From the fourth week of life, specific receptors for 1,25(OH)2D3 were predominant. In the third week after birth, both the receptor for 1,25(OH)2D3 and the 24,25(OH)2D3 binding protein were present. We have used a sensitive parameter for vitamin D action, the stimulation of creatine kinase BB (CKBB) activity, to measure the response of kidneys from vitamin D-deficient or normal rats. In the first days of life of vitamin D-deficient rats, the kidneys did not respond to either vitamin D metabolite; in the second week of life, there was stimulation of renal CKBB only by 24R,25(OH)2D3; beginning in the fourth week of life, only 1,25(OH)2D3 stimulated renal CKBB. However, during the third week of life, CKBB activity was increased by both metabolites. In normal animals, which showed a lower CK activity at all ages, the response was similar to that in vitamin D-deficient animals but the peak was achieved a few days later. The stimulation of CKBB by vitamin D metabolites occurred in all the zones of the kidneys. An increase in renal CKBB by 1,25(OH)2D3 was also detected immunohistochemically. The increase of CKBB activity caused by the two vitamin D metabolites at different stages of development, closely correlated with changes in the presence of the 1,25(OH)2D3 receptor or the 24,25(OH)2D3 binding protein, suggests a specific role for each metabolite during renal development.

Autoradiographic localization of 24R,25-dihydroxyvitamin D3 in epiphyseal cartilage.

There is emerging evidence for specific binding sites and biologic action for 24,25(OH)2D3 in the epiphyseal cartilage. The present study was undertaken to identify the target cells of 24,25(OH)2D3 in the epiphyses of rat bone using an autoradiographic technique. Pieces of epiphyseal cartilage obtained from 4-day-old vitamin D-deficient rats were incubated for 15 or 60 min with [3H]-24,25(OH)2D3 in the presence or absence of 100-fold excess of 25(OH)D3, 1,25(OH)2D3, or 24R,25(OH)2D3. The pieces were prepared for autoradiographic study by a new modified fixation method. Cytoplasmic and nuclear concentration of radioactivity was observed in all cell layers of the epiphyseal cartilage except for the hypertrophic cartilage zone. The highest concentration of radioactivity was seen in the proliferating chondroblasts of the columnar zone. After 15 min of incubation the radioactivity was seen mainly in the cell membrane and cytoplasm, whereas at 60 min radioactivity was also prominent in the nuclei. The competition with excess of cold metabolites revealed that only 24R,25(OH)2D3 caused a significant decrease in cytoplasmic and nuclear radioactivity. These data support the biochemical studies showing that the epiphyseal cartilage is a target tissue for 24,25(OH)2D3.

Biochemical change in the liver and kidney of rats following parathyroidectomy.

The effect of hypoparathyroidism and low blood calcium on enzyme levels in rat liver and kidney is shown. Four animal groups were used: parathyroidectomized (PTX), PTX with CaCl2 added in the drinking water, sham-operated controls and sham-operated with CaCl2 added in the drinking water. PTX significantly lowered serum parathyroid hormone (PTH) and calcium. Supplementation of CaCl2 in the drinking water increased serum Ca levels in PTX rats but not in the controls. Significant changes in several liver and kidney enzymes were seen. Most affected were the liver NADP dependent enzymes, glucose-6-phosphate dehydrogenase and malic enzyme. Similar patterns but with relatively smaller changes were seen in the liver enzymes, lactic dehydrogenase, hexokinase, and aspartate transferase. No significant differences between the groups were seen in the levels of malic dehydrogenase, isocitric dehydrogenase, fructose-6-phosphate kinase and cholinesterase. In the kidney, which was less affected than the liver, the only significant difference was seen in the level of malic enzyme. Serum total lipids in the PTX group were significantly lower. All the changes seen were partially reversed by Ca supplementation in the drinking water.

Effect of parathyroid hormone and uremic sera on the autoagglutination and sedimentation of human red blood cells.

Parathyroid hormone (PTH) caused a dramatic acceleration of erythrocyte sedimentation rate (ESR). This effect was calcium dependent and was partially reversed by verapamil. It was not mimicked by 5 mumol/l calcium ionophore A-23187. Following the removal of PTH from the cell suspension the ESR returned to normal. PTH also caused haemagglutination, the reaction was Ca2+ dependent, pH dependent and was partially reversed by verapamil. High levels of Ca2+ ionophore A-23187 mimicked this phenomenon. Magnesium ions even at concentrations of 5 mmol/l did not replace Ca2+, while Ca2+ at concentrations of 3 mmol/l and above caused haemagglutination. The glycolytic inhibitor NaF at levels of 1 mmol/l did not inhibit haemagglutination. The polyamines pertusin and spermidin, prostaglandins PGE2 and PGF, and the calcium hormone calcitonin, did not reproduce the PTH effect. Dialysate from serum of patients with chronic renal failure and hyperparathyroidism caused haemagglutination, while dialysate from patients with chronic renal failure following parathyroidectomy and normal individuals did not cause this phenomenon. It seems that abnormal erythrocyte behaviour seen in patients with chronic renal failure is caused by PTH which leads to modified Ca2+ metabolism in these cells.