Simple, Rapid Enzyme-Linked Immunosorbent Assay (ELISA) for the Determination of Rat Osteocalcin

Determination of the concentration of osteocalcin in rat serum is frequently performed using a commercially available radioimmunoassay (RIA). However, this assay takes 3 days to complete, uses radioactive material, and has a narrow linear range. The limited range of the RIA makes it necessary to test multiple dilutions of the sample which frequently results in values that differ, depending on the dilution. In order to overcome these limitations, we have developed an ELISA that utilizes the same standards and anti-rat osteocalcin antiserum, as is used in the RIA. The principle of the ELISA is that the osteocalcin in the sample competes with osteocalcin previously immobilized on a microtiter plate to bind to the available anti-rat osteocalcin antibodies. The amount of antibody bound to the immobilized osteocalcin is determined colorimetrically using a secondary antibody coupled to alkaline phosphatase. This ELISA has a three-log linear response with a sensitivity of 0.1–0.15 ng/ml and intra- and interassay coefficient of variance (CV) values of less than 10%. Most importantly, the assay is rapid and only requires a 2-hour incubation of the sample with the antiserum. The incubation time is important since we and others have observed a significant decrease in the osteocalcin level from serum samples incubated for long periods of time with the antiserum, presumably due to degradation of the osteocalcin. In general, the commercially available RIA gives osteocalcin values that are one-half to one-fourth that of the ELISA because the RIA requires a 48-hour incubation time. Received: 14 November 1997 / Accepted: 9 July 1998     

High Bilirubin Levels Interfere with Serum Tartrate-Resistant Acid Phosphatase Determination: Relevance as a Marker of Bone Resorption in Jaundiced Patients

Serum tartrate-resistant acid phosphatase (TRAcP) activity is considered to be a biochemical marker of bone resorption. Recently, a lack of specificity of collagen-related markers for assessing bone turnover has been observed in patients with chronic liver disease. Thus, it could be of great interest to determine serum TRAcP activity in such patients. However, nonspecificity of the analytical reaction could occur when hemolyzed, lipemic, or icteric specimens are analyzed. Therefore, we have studied the interference caused by bilirubin in the measurement of serum TRAcP activity using the Hillmann method. The interference was assessed in two pools of serum containing different bilirubin concentrations but with similar total AcP levels. Mixing proportional parts of the two pools, 10 samples were also obtained. Serum activities of total AcP and TRAcP, and the concentration of bilirubin were measured in the 10 samples. Both the actual and the expected values obtained by theoretical calculations were compared. Serum bilirubin values of 2.4 mg/dl showed a negative interference of 15% in the determination of serum TRAcP activity, whereas values of bilirubin higher than 10 mg/dl interfered totally with the measurement of serum TRAcP. Bilirubin did not interfere with the total AcP determination. This study clearly shows the interference of bilirubin in the determination of serum TRAcP. This finding should be considered when bone metabolism disorders are evaluated in jaundiced patients. Received: 6 April 1998 / Accepted: 1 October 1998     

Radiation doses of yttrium-90 citrate and yttrium-90 EDTMP as determined via analogous yttrium-86 complexes and positron emission tomography

Yttrium-90 is used for palliative therapy for the treatment of skeletal metastases, but because it is a pure - emitter, data on the pharmacokinetics and radiation doses to metastases and unaffected organs are lacking. To obtain such data, the present study employed yttrium-86 as a substitute for⁹⁰Y, with detection by positron emission tomography (PET). The study compared the properties of two different⁸⁶Y complexes —⁸⁶y-citrate and⁸⁶Y -ethylene diamine tetramethylene phosphonate (EDTMP) — in ten patients with prostatic cancer who had developed multiple bone metastases (the ten patients being divided into two groups of five). Early dynamics were measured up to 1 h post injection (p.i.) over the liver region, followed by subsequent whole-body PET scans up to 3 days p.i. Absolute uptake data were determined for normal bone, bone metastases, liver and kidney. Radiation doses were calculated according to the MIRD recommendations. Based on the pharmacokinetic measurements of the distribution of the⁸⁶Y complexes, it was possible to calculate radiation doses for the bone metastases and the red bone marrow delivered by complexes containing⁹⁰Y. In 1 cm³ of bone metastasis, doses of 26±11 mGy/MBq and 18±2 mGy/MBq were determined per MBq of injected⁹⁰Y- citrate and⁹⁰Y- EDTMP, respectively. The doses to the bone marrow were 2.5±0.4 mGy/MBq for⁹⁰Y- citrate and 1.8±0.6 mGy/MBq for⁹⁰Y-EDTMP.⁸⁶Y and PET provide quantitative information applicable to the clinical use of⁹⁰Y. This method may also be useful for the design of other⁹⁰Y radiopharmaceuticals and for planning radiotherapy dosages.     

Ipriflavone does not alter bone apatite crystal structure in adult male rats

We have previously found that a short-term treatment with high doses of ipriflavone increased bone density and improved the biomechanical properties of adult male rat bones, without altering their mineral composition. To determine whether this effect can be associated with alterations of bone crystal structure, we have performed X-ray diffraction analysis of bones obtained from rats treated with ipriflavone at doses that were effective in inducing favorable changes on bone density and biomechanics. Eighteen-week-old male Sprague Dawley rats were treated by oral route with either ipriflavone (200 or 400 mg/kg/day), or its vehicle for 12 weeks. The treatment was well tolerated and body weight increased to the same extent in all animals. As a measure of bone crystallinity, we examined the (310) and (002) reflections of the X-ray diffraction patterns, corresponding to the directions perpendicular and parallel to the c-axis of the crystals, respectively. No major differences were observed between ipriflavone-treated and control animals for the broadening parameter 1/2 for (310) and (002) peaks, as well as for lattice parameters. Therefore, a 12-week treatment with ipriflavone at high doses does not induce significant modifications of bone crystallinity. Thus. the positive effect of ipriflavone on bone mineral density appears to be associated with an increased apatite crystal formation rather than an increase of crystal size. These results provide further evidence for the safety and usefulness of ipriflavone in the treatment of osteoporotic syndromes.     

Effects of vitamin D3 metabolites on bone cell calcium transport

Summary The vitamin D₃ metabolite, 25-hydroxycholecalciferol, at concentrations of 0.01 to 10.0 g/ml, decreased calcium uptake by isolated bone cells. The effect occurred within 1 min after the simultaneous addition of metabolite and⁴⁵Ca. Lactic acid and ATP production by the cells was not affected. 24(R), 25-dihydroxycholecalciferol produced a similar decrease in calcium uptake. Vitamin D₃ had no effect at concentrations from 0.01 to 10.0 g/ml. No effect of 1,25-dihydroxycholecalciferol on calcium uptake was observed with concentrations from 0.1 to 100 ng/ml and various preincubation periods extending to 2 h. None of the agents had any effect on calcium efflux. The effects of 25-hydroxycholecalciferol and 24(R), 25-dihydroxycholecalciferol on calcium uptake were not seen in isolated fetal rat skin cell preparations.     

The vascular ‘sunburst’ appearance of osteosarcoma: A new angiographic finding

The angiographic analogue of the sunburst, (right angle) periosteal new bone formation in osteogenic sarcoma is described. The angiographic findings in this tumor and their relationship to the pathologic appearance are discussed.     

Greffe Intertibio-péronière dans le Traitement des Pseudarthroses Fistuleuses Diaphysaires de Jambe

Résumé Les auteurs présentent une série de 30 pseudarthroses fistuleuses de jambe traitées par greffe intertibiopéronière, revues avec un recul allant de 3 à 9 ans.La voie d'abord utilisée pour la greffe est le plus souvent rétropéronière; dans 14 cas l'immobilisation a été réalisée par un plâtre et dans 16 cas par un fixateur externe (chez 9 malades, l'opération a été réalisée après mise en place du fixateur). 28 guérisons sont constatées (25 rapides en 3 à 8 mois, 3 plus lentes) et 2 échecs.Les auteurs insistent sur l'intérêt de la voie rétropéronière qui permet le plus souvent de rester à distance des parties molles «septiques» et du fixateur externe comme moyen de contention.L'indication de cette méthode concerne surtout les pseudarthroses infectées anciennes avec des extrémités osseuses atrophiques ou en cas de perte de substance osseuse. Dans les autres cas, la décortication de Judet est plus simple et permet d'obtenir d'excellents résultats.

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Intertibio-peroneal graft in the treatment of infected non-united legs

Summary The authors present a series of 30 cases of fistular non united fractures of the leg treated by interosseous bone graft. Survey was performed from 3 to 9 years.In most cases, the surgical approach is made behind the fibula; in 14 cases, immobilization by means of a plaster cast and in 16 cases by means of external fixation (in 9 cases, external fixation bar + screws was realised first).Healing occured in 28 cases (quick in 25 cases, i.e. within 3–8 months, slow in 3 cases) and 2 failures.The authors emphasize the value of the posterior (retrofibular) approach, which avoids the distal septic soft areas, and of the bar-screws device.The procedure is mostly relevant in old infected non united fractures, with atrophic bony extremities or with boneloss.In the other cases, Judet's procedure is more simple and allows excellent results.

     

Comparative assessment of bone mineral density of the forearm using single photon and dual X-ray absorptiometry

Summary Forearm bone mineral density (BMD) was measured at proximal and distal sites by ¹²⁵I single photon absorptiometry (SPA) and by dual energy X-ray absorptiometry (DXA) in 67 consecutive subjects, aged 18–75 years. Correlations and regression equations between these two techniques were determined. All forearm measurements were significantly correlated with each other (r=0.599–0.926; P0.0001). Although SPA and DXA correct for fat in different ways, we found similar correlation and regression equations in women with body mass index measurements above and below the mean. In addition, forearm measurements by both techniques were moderately correlated with vertebral spine and hip BMD. We conclude that overall, SPA forearm measurements in a population can be calibrated to DXA measurements if necessary, and that DXA forearm measurements are as predictive of the remainder of the skeleton as SPA measurements.     

Effect of a five-week swimming program on rat bone: A histomorphometric study

Summary To specify the exercise-induced changes on different skeletal sites, the effect of a 5-week endurance swin training was studied in rats. Eighteen Lyon strain (Sprague-Dawley) 5-week old female rats were divided into nine sedentary and nine swimming rats. Each swim training session was increased by 15 minutes from 2–6 hours per day. A histomorphometric study was performed at the primary and secondary spongiosa of the distal femur and at the secondary spongiosa of lumbar and thoracic vertebral bodies. After training, bone loss was observed in the secondary spongiosa of lumbar vertebral bodies (24.7%) and in the primary spongiosa of distal femur (15.2%). A tendency to bone loss was also detected in the secondary spongiosa of distal femur (10.8%), whereas no change was detected in thoracic vertebral bodies. In secondary spongiosa, bone loss was accompanied with a thinning of trabeculae. Total eroded surfaces and osteoid surfaces were significantly decreased in the three studied skeletal sites, suggesting a decreased bone turnover. The decreased thickness of osteoid seams in both lumbar vertebrae and distal femur could mean that the osteoblastic activity has also been altered at the cell level, leading to thinning of trabeculae. Five-week swim training with such duration and intensity of exercise appears unable to increase bone volume in rats and, therefore, causes adverse effects. The three studied bones seemed to adapt differently to experimental conditions. The lack of ground reaction forces induced by water immersion might have contributed to the observed bone loss. Normal gravity would be an important cofactor in the osteogenic effects of exercise.     

Competitive adsorption of magnesium and calcium ions onto synthetic and biological apatites

Summary Magnesium (Mg) is a conspicuous constituent of hard tissues but its possible role in biomineralization is poorly understood. It is possible that Mg²⁺ adsorbed onto bioapatites may contribute to the modulation of crystal growth as such inhibitory activity has been reported for synthetic apatites. The present study was undertaken to determine the adsorption isotherms of Mg ions onto synthetic apatites and biominerals in tooth and bone tissues in the presence of other ions of natural occurrence. Synthetic crystals used as adsorbents were hydroxyapatite and, as a better prototype for the biomineral, Mg-containing carbonatoapatite. Human enamel and dentin materials were obtained from extracted, caries-free, permanent teeth. Porcine dentin materials at two developmental stages were obtained from erupted deciduous and unerupted permanent teeth of a 6-month-old slaughtered piglet. Porcine bone was obtained from the cortical portion of the mandible of the same animal. All biomineral samples were pulverized and then treated by plasma ashing (deproteination) at about 60°C. Each of the powdered samples was equilibrated in solutions containing various initial concentrations of Mg²⁺, Ca²⁺, and Na⁺ (or K⁺) as nitrate salts. Following equilibration, concentrations (and activities) of magnesium and calcium ions in the experimental solution were determined. The pH values of the equilibrium solutions were in the range of 6.2–6.5. Experimental data of the Mg adsorption onto hydroxyapatite were interpreted on the basis of a Langmuir-type model for binary systems assuming competition of Mg²⁺ and Ca²⁺ for the same adsorption sites on the crystal surfaces of the apatites. According to this model, the adsorbed Mg is expressed as a function of the ionic activity ratio (Mg²⁺)/(Ca²⁺) in the equilibrium solution. The model contains two parameters, the adsorption selectivity constant K_s and the maximum number of adsorption sites N (mol/g). The numerical values of K_s were similar for all adsorbents used (synthetic and biological) and indicated the preferential adsorption of Ca²⁺ probably due to spacial restrictions extending to the very surface of the crystals. The initial level of Mg²⁺ in the surface pool was different in the various biominerals, probably reflecting the composition of fluid in which the biominerals were formed. Whereas the surface pool of Mg of human enamel was marginal, only 5% of the total Mg, significant fractions of the total Mg in human and porcine dentins (about 20–30%), and porcine bone (about 40%) existed on the crystal surfaces. There were significant differences in the total Mg and the value of the parameter N between young (unerupted) and mature (erupted) dentin minerals. It was ascertained that the occupancy of adsorption sites by Mg ions became greater with maturation of the dentin tissues. The overall results suggest that the Mg-mineral interaction in tooth and bone tissues may be a highly tissue-specific process, presumably reflecting differences in fluid composition (particularly Ca and Mg activities) responsible for biomineralization.