Calcium-41: a technology for monitoring changes in bone mineral.

The rare, long-lived radiotracer, 41Ca, measured by accelerator mass spectrometry in the urine or serum following incorporation into the bone provides an ultra-sensitive tool to assess changes in bone calcium balance in response to an intervention. Changes in bone balance can be followed for years with one small dose that is both radiologically and biologically non-invasive. Sequential interventions can be compared, with greater precision than they can with biochemical markers of bone turnover and with greater power than with bone densitometry. This method is especially useful to screen interventions over a period of weeks. The development and validation of this tool and its applications are reviewed. Mini abstract: Use of 41Ca measured in the urine or blood by accelerator mass spectrometry to assess bone balance provides a tool to compare the relative efficacy of multiple interventions. This perspective provides insights in the use of this novel method and comparisons with more traditional methods for evaluating the efficacy of interventions.

Bone seeking labels as markers for bone turnover: effect of dosing schedule on labeling various bone sites in rats.

Urinary excretion of bone labels can be used to monitor bone resorption. Here we investigate the effects of dosing frequency on label incorporation of various sites when bone turnover was perturbed by ovariectomy. We compared tritiated tetracycline ((3)H-TC) and (45)Ca in two studies. Nine-month-old rats were given single or multiple injections of (3)H-TC and (45)Ca and sacrificed after 7 or 14 days. Six-month-old OVX rats were given (3)H-TC and (41)Ca tracers 1 or 3 months following ovariectomy (OVX + 1 mo or OVX + 3 mo, when bone turnover was higher or lower, respectively) and sacrificed 1 week, 1 month, 3 months, or 6 months postdose. Twenty-four-hour urine pools over 2-4 consecutive days as well as the proximal tibia, femur midshaft, lumbar vertebrae (L1-L4), and remaining skeleton were analyzed for (3)H, (45)Ca, and calcium content. Bone turnover as assessed by urinary (3)H-TC was greater in OVX + 1 mo compared to OVX + 3 mo rats up to 6 months postdose. (45)Ca labeling efficiency (% dose/g Ca) was significantly higher than for (3)H and labeling was higher in trabecular-rich than cortical-rich bone. This study affirms that a single administration of either (3)H-TC or (45)Ca is a useful approach to measuring bone turnover directly. The amount of label incorporation into bone was greater in bone sites that were more metabolically active and in all sites when closer vs farther from OVX.

Variability of calcium absorption.

Variability in calcium absorption was estimated in three groups of normal subjects in whom Ca absorption was measured by standard isotopic-tracer methods at interstudy intervals ranging from 1 to 4 mo. Fifty absorption tests were performed in 22 subjects. Each was done in the morning after an overnight fast with an identical standard breakfast containing a Ca load of approximately 250 mg. Individual fractional absorption values were normalized to permit pooling of the data. The coefficient of variation (CVs) for absorption for the three groups ranged from 10.57 to 12.79% with the size of the CV increasing with interstudy duration. One other published study presenting replicate absorption values was analyzed in a similar fashion and was found to have a CV of absorption of 9.78%. From these data we estimate that when the standard double-isotope method is used to measure Ca absorption there is approximately 10% variability around any given absorption value within an individual human subject and that roughly two-thirds of this represents real biological variability in absorption.

Determination of the retention of 47Ca by whole-body counting.

Retention of intravenously or orally administered 47Ca in the human body are described by a two-parameter function. It is then sufficient to make only a few whole-body measurements to determine the retention function, avoiding faeces sampling and stool markers. Seven days after intake the non-absorbed calcium was excreted and the model agreed with the measured relative retention. Absorption of calcium could then, in some cases (e.g. comparative studies), be described by relative retention at the 7th day after intake.

Determining the relative toxicity and RBE of internal emitters in animals.

It is almost impossible to conduct a perfect study of the relative toxicity of the radiations produced by different radionuclides. This is because the results of such studies are commonly confounded by spatial and temporal differences in the distributions of dose produced by the radionuclides employed. In addition, the results of a study designed to overcome these problems (using matched radionuclides incorporated within fused clay particles) revealed additional characteristics of an ideal study. These included the use of sufficient numbers of animals to give the study statistical power; the derivation of all causes of death and of survival for the analysis; the use of relative risk, rather than crude incidence data, to determine toxicity ratios; the cautious use of relative biological effectiveness values derived from fitted curves; and the preferred use of relative toxicity values derived directly from the data.

The measurement of calcium absorption and absorption rate with an external arm radioactivity counter.

1. A large-volume scintillation counter was used to measure calcium absorption from the ratio of forearm uptake of 47Ca after oral 47CaCl2 (administered with milk) to forearm uptake after intravenously administered 47CaCl2. 2. In some subjects serial measurements of both forearm uptake of 47Ca and blood 47Ca radioactivity were also recorded, and by using deconvolution both total calcium absorption and calcium absorption rate were determined. 3. The forearm ratio determination of 47Ca absorption correlated well with that obtained by deconvolution of either serial blood 47Ca or forearm 47Ca measurements provided that the forearm radioactivity measurements were made at least 8 h after the administration of 47CaCL2. 4. Although the two deconvolution techniques gave similar estimates of total calcium absorption there were discrepancies between their measurements of calcium absorption rate. These discrepancies were reduced but not eliminated by the use of additional lead shielding around the Armac counter.

[Evaluation of intestinal absorption of calcium by means of double-isotope methods and oral administration of the tracer]. / Valutazione dell'assorbimento intestinale del calcio me diante i metodi del doppio isotopo e la somministrazione orale del tracciante.

The percent intestinal absorption of calcium was measured in normal volunteers and in patients with idiopathic hypercalciuria employing the deconvolution method, the ratio of the two administered isotopes at equilibrium and the percent of dose present in plasma 2 hr after oral administration of the tracer. Comparison of results obtained showed that the technique based on the ratio between the two radioisotopes overestimates intestinal absorption by about 9% with respect to values calculated with the deconvolution method, but gives results comparable to those determined by oral administration of the isotope. The percent dose of the tracer 2 h after i.v. administration is closely correlated with the size of the miscible calcium pool. A less significant correlation exists between the size of the pool and percent of the dose 2 h after oral administration.

Estimation of true calcium absorption.

Measurements of true calcium absorption fraction in women were evaluated to see how closely calculations based on a single measurement of serum specific activity after administration of an oral calcium tracer might approximate the absorption value derived from the full, double-isotope absorption procedure. True absorption, body size, and miscible pool turnover could together explain better than 93% of the variance in serum calcium specific activity values 5 hours after a tracer-labeled test meal. Because measurement of pool turnover is not available routinely, it was dropped from the model, and a predictor equation was developed that allowed estimation of true absorption from the 5-hour serum specific activity value, height, and weight. These variables explained 90.8% of the variance in the 5-hour values and gave estimates of true absorption with a 95% confidence interval of +/- 0.055. This small range of uncertainty makes the procedure useful in estimating absorption efficiency for calcium therapy in routine clinical practice.

A comparative study of two methods of measurement of fractional calcium absorption: results in normal individuals and patients with various disturbances of calcium metabolism.

Two methods of measurement of fractional calcium absorption have been compared in the same individuals. There is a significant correlation between fractional 47Ca absorption as measured with a blood counting method and with an external counting method after intravenous and oral administration of 47Ca (P less than 0.001). Two varieties of the external counting method, namely with intervals of 2 h and 7 days between the i.v. and oral doses, have been compared. In a group of twenty-one patients with chronic renal failure (not on haemodialysis), 47Ca absorption as measured by external counting was significantly lower than in the control group (P less than 0.001). When put on a dialysis programme a group of six patients showed a significant increase of the fractional 47Ca absorption (P less than 0.01).

Accelerating effect of chitosan intake on urinary calcium excretion by rats.

The effect of chitosan on calcium (47Ca) metabolism was investigated in rats. The whole-body retention of 47Ca by rats fed on a 5% chitosan diet was significantly decreased when compared with that of rats fed on a cellulose diet, but showed no significant difference from that of rats fed on a fiber-free diet. Although there was no significant difference in the fecal excretion of 47Ca between the chitosan group and the cellulose or fiber-free group, the urinary excretion of 47Ca was significantly increased in the chitosan group when compared with the cellulose group. These results suggest that dietary chitosan would affect the calcium metabolism in animals.

Effects of low-dose-rate beta-irradiation on vascular smooth muscle cells: comparison with high-dose-rate exposure.

PURPOSE: Radiation therapy is undergoing extensive preclinical and clinical testing as a new tool to reduce restenosis after vessel injury. To date, however, no definite dose threshold has been identified after radioactive stent implantation. In this study, we compared the in vitro response of pig vascular smooth muscle cells (SMC) to conventional high-dose-rate (HDR) irradiation with the response to continuous low-dose rate (LDR) that could result from exposure to a radioactive stent. MATERIALS AND METHODS: Catheter-based radiotherapy delivers single doses at HDR whereas radioactive stents use a continuous LDR approach. Single doses in excess of 10 Gy have clearly shown a reduction in neointima formation and negative vessel remodeling in several animal models. Because dose rate is an important parameter modulating the overall biological response to ionizing radiation, we have compared the in vitro response of pig aortic SMC at conventional HDR (1.5 Gy/min) and at LDR (0.675 Gy/h). RESULTS: SMC showed significant repair of sublethal DNA damage and about twice the dose was necessary at the LDR to produce the same effect as that seen at the HDR. CONCLUSION: In vitro SMC exhibit a significant dose-rate effect that indicates that radioactive stents could deliver the dose at a sufficiently high dose rate to compensate for cell proliferation while at the same time the total dose should be increased to account for sublethal damage repair. This finding has important implications for the design of a radioactive stent.

Effect of fasting and parathyroid hormone injection on plasma 45Ca concentrations in rats.

Young male rats were administered 45Ca 5 days to 2 weeks prior to use. All rats were either parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) and given several days to recover from surgery. The first group of rats were maintained on a 12 h dark-fed and 12 h light-fasted daily cycle. The remainder of the rats were used for parathyroid hormone (PTH) studies (0.1-0.6U/g body weight) following which blood samples were obtained from the tail for 1 to 6 h. Two groups of these rats were bilaterally nephrectomized 18 h before PTH injection. Two contrasting results were obtained: in PTX (or TPTX) rats maintained on the closely regulated food and light regime, plasma 45Ca concentrations rose markedly each day at the start of the fasting period and then fell slowly. Total plasma calcium values fell throughout the fasting period. A similar rise and fall was also observed in 45Ca values of rats experimentally fasted after being maintained with food continuously available. In contrast, in all PTX or TPTX rats, PTH injections was followed by an equal rise in both plasma calcium and 45Ca values so that for the first few hours plasma 45Ca specific acitvity was unchanged. These data are consistent with the concept of a bone fluid compartment (BFC) separated by a cellular interface from the primary extracellular fluid space (ecf). It is postulated that through this cellular interface calcium is actively "pumped" from the BFC to the ECF. The rise in plasma 45Ca values at the start of fasting is explained on the basis of decreased entry of stable calcium from the gastrointestinal tract and a continued movement of calcium and 45Ca from the BFC to the ECF. The concomitant increase in plasma calcium and 45Ca during the first few hours after PTH injection is explained by a rapid action of PTH to increase the rate of calcium movement from BFC to ECF by its action at the cellular interface, without altering 45Ca specific activity until such time as dissolution of bone crystals is required as a supply of calcium.

Comparison between the fractional isotopic calcium absorption and an oral calcium tolerance test.

In 27 subjects with several disorders of calcium metabolism, the fractional intestinal absorption of 47CaCl2 was rather poorly correlated with the urinary output of calcium or with the maximal increase of serum calcium after an oral calcium load. Conversely, a good correlation was observed with the product of these parameters. We propose that this product be used as an estimate of intestinal calcium absorption when a radioisotopic method is not available.

[The oral Ca47 test and cynamics of Ca47: comparative evaluation in the study of the digestive absorption of calcium in man]. / Il test orale al Ca-47 e la dinamica del Ca-47: valutazione comparativa nello studio dell'assorbimento digestivo del calcio nell'uomo

The intestinal absorption of calcium is one of the most important processes regulating calcium metabolism. Its measurement requires the estimation of dietary and faecal calcium over a period of one week, but this procedure is frequently liable to error. The availability of two isotopic procedures contributed to the successful pursuit of measuring intestinal absorption of calcium. In the first procedure the indirect assessment of calcium absorption is based on the kinetic study of plasma radioactivity curve after an intravenous dose of radiocaldium at the same time with a calcium balance study over a period of 6 days, In the second isotopic procedure the calcium absorption is directly calculated from the plasma radioactivity and residual faecal activity following an oral dose of radioactive calcium. We have investigated by the two methods 25 patients (one normal subject and 24 affected by a variety of clinical disorders of calcium metabolism). The excellent agreement, statistically significant, between the data obtained by the two methods suggests that both procedures are adequate to assess intestinal absorption of calcium in man.

[The effect of ultrasonic oscillations of different frequencies on radionuclide accumulation in the eye tissues]. / Vliianie ul'trazvukovykh kolebanii razlichnoi chastoty na nakoplenie radionuklidov v tkaniakh glaza.

The paper describes results of experimental investigations in 25 rabbits for the purpose to study permeability of eye tissues under the action of ultrasound of different frequencies (127, 295, 470, 660, 880, 1000, 2750 kHz). The following radioactive nuclides were used: 99mTc--pertechnetate, 125I in a form of KI and 45CaCl2. In investigations with 99mTc--pertechnetate and 45CaCl2, their accumulation in the aqueous humor, vitreous body, cornea, iris, sclera, crystalline lens, choroid and retina was studied. It was found that permeability of eye tissues depends, to a great extent, on ultrasound frequency. The highest amount of radionuclides penetrate into the eye media and eye tissues under the action of ultrasound with 470, 660, 880 kHz frequencies. With increase or decrease of ultrasound frequency the permeability decreases. This fact should be considered when applying phonophoresis.

The effect of dietary calcium on bone metabolism in young and aged female rats using a short-term in vivo model.

The use of high dietary calcium supplementation in the treatment of patients with osteoporosis is controversial. The present study examined the mechanisms underlying the effects of calcium supplementation by investigating the influence of dietary calcium on bone dynamics in young and aged rats. A 2 x 2 x 2 factorial design was utilized with 0.2% (low) or 1.0% (high) calcium, 2- or 24-m-old female Long-Evans rats that were implanted subcutaneously with demineralized (DB) and mineralized (MB) bone powder. The four groups of rats were fed each of the respective diets for 11 wk and then implanted with one #5 gelatin capsule containing 30 mg of DB and another containing 100 mg of MB powder. The animals were injected intraperitoneally with 0.1 microCi/g body weight with 45Ca 14 h before the end of experiment. The ectopic bone as well as the right femurs were harvested 14 d after the rats were implanted. Marker enzyme activities (alkaline-formation and acid-resorption phosphatase), 45Ca uptake and calcium content were measured in the implants and the distal epiphyses of the right femurs. Bone turnover was higher in the young rats than in the old animals, and high dietary calcium in the young animals increased bone formation, as indicated by alkaline phosphatase activity. Dietary calcium level did not affect ectopic bone formation or resorption in the aged rats. The results indicate that high dietary intake of calcium does not affect bone dynamics in aged female rats but does increase bone formation in young rats.