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.

Effect of Hesperidin With and Without a Calcium (Calcilock) Supplement on Bone Health in Postmenopausal Women.

CONTEXT: Citrus fruits contain unique flavanones. One of the most abundant of the flavanones, hesperidin, has been shown to prevent bone loss in ovariectomized rats. OBJECTIVE: The objective of the study was to measure the effect of hesperidin with or without calcium supplementation on bone calcium retention in postmenopausal women. DESIGN: The study was a double-blind, placebo-controlled, randomized-order crossover design of 500 g hesperidin with or without 500 mg calcium supplement in 12 healthy postmenopausal women. Bone calcium retention was determined from urinary excretion of the rare isotope, (41)Ca, from bone. RESULTS: Calcium plus hesperidin, but not hesperidin alone, improved bone calcium retention by 5.5% (P < .04). CONCLUSION: Calcium supplementation (Calcilock), in combination with hesperidin, is effective at preserving bone in postmenopausal women.

Validation of urinary calcium isotope excretion from bone for screening anabolic therapies for osteoporosis.

SUMMARY: Urinary excretion of calcium tracers in labeled individuals decreases in response to antiresorptive therapy, providing a tool to rapidly screen potential therapies. Using teriparatide, we demonstrate in this study that anabolic therapy also decreases tracer excretion, confirming that this method can also be used to screen potential anabolic therapies. INTRODUCTION: Changes in urinary excretion of calcium tracers from a labeled skeleton may be a rapid and sensitive method to screen potential therapies for osteoporosis. This method has been used to screen antiresorptive therapies, but the effect of anabolic therapies on tracer excretion is unknown. METHODS: Eight-month-old female Sprague Dawley rats (n = 11) were given 50 µCi (45)Ca iv. After a 1-month equilibration period, baseline urinary (45)Ca excretion and total bone mineral content (BMC) were measured. Rats were then treated with 30 µg/kg teriparatide sc per day, a bone anabolic agent, for 80 days. Urine was collected throughout the study and analyzed for (45)Ca and total Ca, and BMC was measured at the beginning and end of the study. RESULTS: Teriparatide decreased urinary (45)Ca excretion by 52.1 % and increased BMC by 21.7 %. The change in bone calcium retention as determined by the ratio of (45)Ca to total Ca excretion in urine from day 6 through 15 of teriparatide treatment was significantly correlated (p = 0.036) with the change in BMC after 80 days of teriparatide treatment. CONCLUSION: Urinary excretion of calcium tracers from labeled bone is an effective method to rapidly screen potential anabolic therapies for osteoporosis.

Calcium isolation from large-volume human urine samples for 41Ca analysis by accelerator mass spectrometry.

Calcium oxalate precipitation is the first step in preparation of biological samples for (41)Ca analysis by accelerator mass spectrometry. A simplified protocol for large-volume human urine samples was characterized, with statistically significant increases in ion current and decreases in interference. This large-volume assay minimizes cost and effort and maximizes time after (41)Ca administration during which human samples, collected over a lifetime, provide (41)Ca:Ca ratios that are significantly above background.

Liquid scintillation based quantitative measurement of dual radioisotopes (3H and 45Ca) in biological samples for bone remodeling studies.

Acute and prolonged bone complications associated with radiation and chemotherapy in cancer survivors underscore the importance of establishing a laboratory-based complementary dual-isotope tool to evaluate short- as well as long-term bone remodeling in an in vivo model. To address this need, a liquid scintillation dual-label method was investigated using different scintillation cocktails for quantitative measurement of (3)H-tetracycline ((3)H-TC) and (45)Ca as markers of bone turnover in mice. Individual samples were prepared over a wide range of known (45)Ca/(3)H activity ratios. Results showed that (45)Ca/(3)H activity ratios determined experimentally by the dual-label method were comparable to the known activity ratios (percentage difference ∼2%), but large variations were found in samples with (45)Ca/(3)H activity ratios in range of 2-10 (percentage difference ∼20-30%). Urine and fecal samples from mice administered with both (3)H-TC and (45)Ca were analyzed with the dual-label method. Positive correlations between (3)H and (45)Ca in urine (R=0.93) and feces (R=0.83) indicate that (3)H-TC and (45)Ca can be interchangeably used to monitor longitudinal in vivo skeletal remodeling.

Interpretation of 41Ca data using compartmental modeling in post-menopausal women.

Calcium-41 (t(1/2) = 10(5) years) can be used after a single dose to follow calcium metabolism over a subject's lifetime. The aims of this study were to expand a (41)Ca kinetic model and estimate bone resorption in women with stable bone loss, compare the rates with those calculated with classical isotope studies, and to use the model to simulate dynamic changes in urinary (41)Ca:Ca ratios and bone balance for the design and interpretation of (41)Ca studies. Forty-two women >5 years post-menopause were given (41)Ca intravenously. Bone mineral content and bone mineral density of total body were measured by dual-energy X-ray absorptiometry at the beginning of the study. Urine collections were made periodically for up to ~5 years while subjects were free living. Urinary (41)Ca:Ca ratios were measured using accelerator mass spectrometry. The isotope data were analyzed by compartmental modeling. Four compartments were necessary to fit the urinary tracer data and total bone calcium. The final model included pathways for absorption, distribution, urinary excretion, and endogenous excretion and was used to calculate rates of bone turnover. Estimates of bone resorption in a subset of the women (n = 13), studied previously in a 3-week balance and full kinetic study with (45)Ca, agreed with those using (41)Ca methodology. Thus, rates of bone resorption can be estimated from (41)Ca urinary data in stable post-menopausal women. The model was used to simulate dynamic changes in urinary (41)Ca:Ca ratios and bone balance, as a result of interventions that perturb calcium metabolism to aid in study design and interpretation.

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.

Evaluation of 41calcium as a new approach to assess changes in bone metabolism: effect of a bisphosphonate intervention in postmenopausal women with low bone mass.

UNLABELLED: A new technique was evaluated to identify changes in bone metabolism directly at high sensitivity through isotopic labeling of bone Ca. Six women with low BMD were labeled with 41Ca up to 700 days and treated for 6 mo with risedronate. Effect of treatment on bone could be identified using 41Ca after 4-8 wk in each individual. INTRODUCTION: Isotopic labeling of bone using 41Ca, a long-living radiotracer, has been proposed as an alternative approach for measuring changes in bone metabolism to overcome current limitations of available techniques. After isotopic labeling of bone, changes in urinary 41Ca excretion reflect changes in bone Ca balance. The aim of this study was to validate this new technique against established measures. Changes in bone Ca balance were induced by giving a bisphosphonate. MATERIALS AND METHODS: Six postmenopausal women with diagnosed osteopenia/osteoporosis received a single oral dose of 100 nCi 41Ca for skeleton labeling. Urinary 41Ca/40Ca isotope ratios were monitored by accelerator mass spectrometry up to 700 days after the labeling process. Subjects received 35 mg risedronate per week for 6 mo. Effect of treatment was monitored using the 41Ca signal in urine and parallel measurements of BMD by DXA and biochemical markers of bone metabolism in urine and blood. RESULTS: Positive response to treatment was confirmed by BMD measurements, which increased for spine by +3.0% (p = 0.01) but not for hip. Bone formation markers decreased by -36% for bone alkaline phosphatase (BALP; p = 0.002) and -59% for procollagen type I propeptides (PINP; p = 0.001). Urinary deoxypyridinoline (DPD) and pyridinoline (PYD) were reduced by -21% (p = 0.019) and -23% (p = 0.009), respectively, whereas serum and urinary carboxy-terminal teleopeptides (CTXs) were reduced by -60% (p = 0.001) and -57.0% (p = 0.001), respectively. Changes in urinary 41Ca excretion paralleled findings for conventional techniques. The urinary 41Ca/40Ca isotope ratio was shifted by -47 +/- 10% by the intervention. Population pharmacokinetic analysis (NONMEM) of the 41Ca data using a linear three-compartment model showed that bisphosphonate treatment reduced Ca transfer rates between the slowly exchanging compartment (bone) and the intermediate fast exchanging compartment by 56% (95% CI: 45-58%). CONCLUSIONS: Isotopic labeling of bone using 41Ca can facilitate human trials in bone research by shortening of intervention periods, lowering subject numbers, and having easier conduct of cross-over studies compared with conventional techniques.

Soy isoflavones do not affect bone resorption in postmenopausal women: a dose-response study using a novel approach with 41Ca.

INTRODUCTION: The purpose of this 3-way crossover study was to identify the effective dose of soy protein isolate enriched with isoflavones for suppressing bone resorption in postmenopausal women using a novel, rapid assessment of antibone resorbing treatments. METHODS: Thirteen postmenopausal women (>or=6 yr since menopause) were predosed with 41Ca iv. After a 200-d baseline period, subjects were given 43 g soy protein/d that contained 0, 97.5, or 135.5 mg total isoflavones in randomized order. The soy protein isolate powder was incorporated into baked products and beverages. Each 50-d intervention phase was preceded by a 50-d pretreatment phase for comparison. Serum isoflavone levels and biochemical markers were measured at the end of each phase. Twenty-four-hour urine samples were collected approximately every 10 d during each phase for 41Ca/Ca analysis by accelerator mass spectrometry. RESULTS: Serum isoflavone levels reflected the amount of isoflavones consumed in a dose-dependent manner. None of the isoflavone levels had a significant effect on biochemical markers of bone turnover, urinary cross-linked N teleopeptides of type I collagen and serum osteocalcin, or bone turnover as assessed by urinary 41Ca/Ca ratios. CONCLUSIONS: Soy protein with isoflavone doses of up to 135.5 mg/d did not suppress bone resorption in postmenopausal women. This is the first efficacy trial using the novel technique of urinary 41Ca excretion from prelabeled bone.

41Ca ultratrace determination with isotopic selectivity > 10(12) by diode-laser-based RIMS.

41Ca ultratrace determination by diode-laser-based resonance ionization mass spectrometry with extremely high isotopic selectivity is presented. Application to environmental dosimetry of nuclear reactor components, to cosmochemical investigations of production cross sections, and biomedical isotope-tracer studies of human calcium kinetics are discussed. Future investigations are possible use in 41Ca-radiodating. Depending on the application, 41Ca isotopic abundances in the range of 10(-9) to 10(-15) relative to the dominant stable isotope 40Ca must be determined. Either double- or triple-resonance optical excitation with narrow-band extended cavity diode lasers and subsequent non-resonant photoionization of calcium in a collimated atomic beam were used. The resulting photoions are detected with a quadrupole mass spectrometer optimized for background reduction and neighboring mass suppression. Applying the full triple-resonance scheme provides a selectivity of approximately 5 x 10(12) in the suppression of neighboring isotopes and > 10(8) for isobars, together with an overall detection efficiency of approximately 5 x 10(-5). Measurements on a variety of sample types are discussed; the accuracy and reproducibility of the resulting 41Ca/40Ca isotope ratios was better than 5%.

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.

Absorption of calcium as the carbonate and citrate salts, with some observations on method.

Calcium supplement use has increased and there is confusion about the relative absorbability of various sources. Absorbability of calcium from the carbonate and citrate salts was compared at 300 mg and 1000 mg calcium loads, ingested as part of a light breakfast meal. Absorption was measured at the high load both by tracer appearance in serum and by the absorptive increment in urinary calcium, and at the low load by the tracer method only. Subjects were 37 healthy adult men and women, studied as outpatients, and each tested on both salts at the same load. Mean tracer absorption (+/- SD) for both salts combined was 36.0% at the 300 mg load and 28.4% at the 1000 mg load. In both experiments the observed mean difference in absorption between salts was very small. By the tracer method the within-subject difference (carbonate less citrate) was +3.3% +/- 1.2% of the ingested dose (mean +/- SEM; P < 0.05) at the high load, and at the low load, 3.6% +/- 2.7% (NS). Combining the two experiments yielded zero difference between sources. By the urinary calcium increment method, the mean difference between salts at the 1000 mg load was 1.8 +/- 4.1 mg (NS). Side-by-side comparisons of the two methods revealed that the tracer method was 3 times more sensitive than the urinary increment method. We conclude that, when taken with food, calcium from the carbonate salt is fully as absorbable as from the citrate, and that the urinary increment method is not sufficiently sensitive to be useful in comparing sources in free-living subjects.

Effects of clomiphene and tamoxifen in vivo on the bone-resorbing effects of parathyroid hormone and of high oral doses of calcitriol (1,25(OH)2D3) in rats with intact ovarian function consuming low calcium diet.

Two experiments were undertaken to study the abilities of clomiphene citrate (20 mg/kg body wt/wk s.c.) and tamoxifen citrate (20 mg/kg body wt/wk s.c.) to slow bone resorption mediated by (a) endogenous parathyroid hormone (PTH) and (b) exogenous calcitriol (1,25(OH)2D3) in vivo in rats with intact ovarian function. Groups of rats with 45Ca-labelled bones were fed a low-calcium (0.01% Ca) diet to stimulate secretion of PTH. Neither clomiphene nor tamoxifen showed the mobilization of 45Ca from femoral bone or prevented the reduction in bone calcium induced by feeding this diet. Moreover these drugs did not depress the urinary excretion of 45Ca or hydroxyproline. These observations indicated that clomiphene and tamoxifen did not inhibit PTH-mediated bone resorption. Administering calcitriol (50 ng/day) orally for 14 days raised plasma calcium, increased urinary 45Ca and its specific activity and decreased femur 45Ca: all these responses were similar in animals receiving calcitriol alone and calcitriol with clomiphene or tamoxifen. The femur 45Ca values (dpm X 10(-3) were: (mean +/- SD, n = 8) placebo, 1901 +/- 127; 1,25(OH)2D3, 1727 +/- 96**; clomiphene + 1,25(OH)2D3, 1694 +/- 93**; tamoxifen + 1,25(OH)2D3, 1664 +/- 61**. (** = P less than 0.01). Thus neither clomiphene nor tamoxifen prevented calcitriol-mediated bone resorption in vivo in the rat.