Competitive protein binding assay for 24,25-dihydroxycholecalciferol

A competitive protein binding assay which measures 24,25-dihydroxycholecalciferol in human serum has been developed using the binding protein from vitamin D-deficient rat kidney. As 25-hydroxycholecalciferol and 25,26-dihydroxycholecalciferol also interact with the binding protein, possible interference by these compounds in the assay has been overcome by preparative chromatography of serum extracts on Sephadex LH 20 prior to assay. The mean serum level of 24,25-dihydroxycholecalciferol in seven normal volunteers was 1.68 ± 0.82 ng/ml whereas patients receiving large therapeutic doses of vitamin D were found to have higher levels. None was detectable in the serum of a vitamin D-deficient patient.     

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).     

Is 24,25-dihydroxycholecalciferol a calcium-regulating hormone in man?

Small doses (1-10 microgram daily) of 24,25-dihydroxycholecalciferol (24,25-(OH)2D3), a renal metabolite of vitamin D of uncertain function, increased intestinal absorption of calcium in normal people and in patients with various disorders or mineral metabolism, including anephric subjects. In five of six patients studied, calcium balance increased, but, unlike 1,25-dihydroxycholecalciferol, 24,25-(OH)2D3 did not increase plasma or urinary calcium concentrations. These results suggest that 24,25-(OH)2D3 may be an important regulator of skeletal metabolism in man with potential value as a therapeutic agent.     

Role of endogenous zinc in bones of newborn rats

Concentration of abundant elements e.g. calcium as well as of elements present in trace amount e.g. zinc in mandibles of 7, 14 an 28 day old newborn rats were determined by X-ray fluorescence analysis. The measurements were carried out by using a measurement system containing X-ray tube ECLIPSE-III and X-ray and gamma ray detector XR-100T-CdTe (Amptek Inc.). Concentration of calcium and zinc depended on the region of interest on the rat's mandible due to mineralization degree conditioned by its function. Increasing age produced a remarkable increase in Ca content in contrast to Zn content in the bone tissue obtained from 7, 14 and 28 day old newborn rats. The calculated Zn/Ca concentration ratio was the biggest for 7 day old newborns and successively decreased with age indicating the important role of zinc at the beginning of bone ontogenesis.     

Blood circulation of long bones in trained growing rats and mice.

The effect of physical training on the blood circulation of long bones was studied in growing rats and mice of NMRI-strain. The animals to be trained and their controls were about 2 weeks old at the beginning of the training. The training took place on a 5 degree inclined treadmill 5 days a week for 3 weeks in experiment I and 7 weeks in experiments II and III. The duration of the daily exercise was progressively increased over 3 weeks. The final exercise bouts were 80 min for moderate and 180 min for intensive training programs. The circulating red cell volume (ml/100 g bone) of the humeral, femoral and tibial bones of the trained animals was lower compared to the controls in all three experiments mainly due to reduced hematocrit values. The circulating blood volume (ml/100 g bone) decreased in the tibial bones of the trained animals in experiment I and showed a decreasing tendency in experiment III, but no significant differences between the groups were observed in the humeral and femoral bones. Yet, when related to the volume of the bones the circulating blood volume (ml/100 ccm bone) was significantly higher in the femoral bones of the trained animals, while the changes in the humeral bones were negligible (experiment III). The results suggest that the vascularity of long bones is affected by physical training. The varying responses in different bones are perhaps due to the amount of mechanical stress during physical activity.     

Effects of 1 alpha(OH)-vitamin D3 and 24,25(OH)2-vitamin D3 on long bones of glucocorticoid-treated rats.

Glucocorticoids may induce osteopenia in experimental animals and in man. In order to study the possible effects of vitamin D metabolites in the prevention of glucocorticoid-induced osteopenia in rats, we administered 1 alpha(OH)-vitamin D3, 24,25(OH)2-vitamin D3 or a combination of both metabolites, by intragastric intubation, to rats treated daily by intramuscular injections of 10 mg/kg cortisone acetate. Treatment with the vitamin D metabolites started after 1 month of glucocorticoid therapy, at the time osteopenia was already present. Cortisone acetate decreased the gain weight, increased alkaline phosphatase (AP) and decreased Ca serum levels. It also decreased tibial wet and ash weight and tibial Ca content. Computerized histomorphometry of sections from the upper tibia showed decreased epiphyseal bone volume and increased bone marrow volume; decreased height of hypertrophic cartilage in the growth plate and decreased amount of persisting cartilage in the metaphyseal bone trabeculae were also observed. Administration of 24,25(OH)2D3 alone did not reduce these glucocorticoid-induced bone changes and sometimes even worsened them. 1 alpha(OH)D3 reversed many of the deleterious effects of cortisone acetate. It reduced serum AP levels, increased serum Ca levels, increased bone ash weight, epiphyseal and metaphyseal bone volume, with a concomitant reduction in epiphyseal and metaphyseal bone marrow volume. The best results were obtained by a combination of 1 alpha(OH)D3 and 24,25(OH)2D3. It is presumed that both metabolites are needed to reduce the impact of glucocorticoids on bone. 1 alpha(OH)2D3 acts on the gut, increasing Ca absorption (which was decreased by glucocorticoids), and 24,25(OH)2D3 directly acts on bone to enhance bone formation and mineralization.     

Triiodothyronine receptors in lymphocytes of newborn and adult rats.

Cytoplasmic and nuclear incorporation of 125I triiodothyronine by thymic lymphocytes has been demonstrated by electron microscopic autoradiography. Incorporation was significantly more pronounced in the newborn than in the adult age. The information emerging from the experime,tal observations contributes further evidence to the more precise interpretation of receptor maturation.     

Evidence for presence of peptide alpha-amidating activity in pancreatic islets from newborn rats.

The peptide alpha-amidating activity of a homogenate of pancreatic islets from 5-7-day-old rats was investigated, using as substrate a glycine-extended tripeptide (D-Tyr-Val-Gly). The islet homogenates had a marked amidating activity, with a Km of 57 microM, a Vmax. of 185 pmol/h per mg and a pH optimum of 7.0. This activity was dependent on the presence of ascorbic acid (in the reduced form) and Cu2+, the optimum concentrations being 4 mM and 40 microM respectively. On fractionation of the homogenate, the highest specific activity was found in the soluble fraction. Exocrine pancreatic tissue showed very low levels of amidating activity.     

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)₂-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, $\text{in}\text{vitro}$. After 40–70 min, 1,25-(OH)₂-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)₂-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)₂-CC to the nucleus, its probable site of action.     

Mechanical properties in long bones of rat osteopetrotic mutations.

Osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. Experimental studies with rat osteopetrotic mutations have shown increased bone density and decreased bone strength. However, it is not known if this reduction in bone strength is only due to changes in structure and geometry or if the tissue properties of bone material itself are changed as well. We have evaluated bone tissue properties with nanoindentation in three osteopetrotic mutations in the rat (incisors-absent ia/ia, osteopetrosis op/op and toothless tl/tl) to test the hypothesis that reduced bone resorption in these mutations results in reduced tissue properties of bone material. No significant differences in elastic modulus or hardness were found between osteopetrotic mutants and their normal littermates (NLMs) in any of the three stocks. This indicates that the tissue properties of bone material are not changed significantly in osteopetrosis, even if the mechanical strength is decreased at the macroscopic level.     

Avian long bones, flight and bipedalism.

Morphological, geometrical, chemical and mechanical characteristics of avian long bones are reviewed. Important differences exist between long bones of birds and mammals. Differences are also present in appendicular bones of birds, either between wing bones and leg bones or proximal (stylopodial) long bones and distal (zeugopodial) long bones. Special emphasis is put on pneumatization, in terms of both phylogenetic origin and geometrical and mechanical characteristics linked to it. Cortical thickness, bending strength and flexural Young's modulus were significantly lower in pneumatized bones than in marrow-filled bones. Possible adaptive reasons for the differences shown are discussed.     

A biomechanical study of the long bones in platyrrhines.

The long bones of 72 individuals of extant platyrrhines, belonging to 17 species (11 genera) were studied by regressions of length, diameters and curvature. Cross-sectional shapes at midshaft and axial and bending strength indicators were also calculated. Results show that forelimb bones scale faster than hindlimb bones, for both length and diameters. Curvature scales faster in the femur than in other bones. Strength indicators showed a high variability in the relative importance of axial and bending loadings. Results are consistent with field observations of locomotor behaviour, mainly as regards quadrupedalism versus suspensory locomotion.     

Investigations of erythropoiesis in newborn rats. Erythropoiesis in newborn rats in physiological conditions.

Erythropoiesis was investigated in suckling rats from the 1st to the 19th day of life when the use of 59Fe. In 2-day and 5-day old rats it was less intensive than in later days. The haemopoietic processes were most intensive between the 7th and 14th day of life. Following this period the activity gradually decreased.