41Ca Tracer Research Articles

A New capability for 41Ca analysis using [formula omitted] at the Xi’an-AMS

The analysis of 41Ca can be quantified by measuring the ratio 41Ca/40Ca. At present, ratios of 41Ca/40Ca ≤ 10−11 can only be measured by accelerator mass spectrometry (AMS). The sensitivity is mainly limited by the interference of the isobar 41K. With compact AMS (≤1 MV), abundance sensitivities of 10−11 to 10−12 have been demonstrated, but this may necessitate the measurement of 39K to correct for the 41K background. With a 3 MV AMS, the dE/dx method can still be used to partially separate 41Ca from 41K. In this work, a new capability for 41Ca analysis has been tested at the Xi’an-AMS. Ca4+ was chosen to achieve a balanced performance between 41Ca/41K separation and transmission efficiency. A 41Ca/40Ca background level of near 10−13 was achieved without the need for 39K correction. This new analytical capability at our facility was then applied to 41Ca tracer samples from our studies on the “White Jade” land snail shells, in which 41Ca was used as a tracer for studying the absorption rate of calcium.

Methodological study on endogenous calcium absorptivity using rats and 41Ca tracing

Elemental calcium plays an important role in human physiology. In order to study the relationship between Ca-intake, Ca-chemical formulation, and Ca-absorptivity, a balance experiment using a 41Ca tracer technique in SD rats was conducted to measure the endogenous fecal calcium and true absorption of calcium. Apparent absorption of calcium was measured as a control to the endogenous calcium labeling experiment. These results show that by using 41Ca labeled endogenous calcium in vivo, researchers could obtain the true calcium absorption data without extrinsic labeling. Therefore, the method was not affected by the chemical structure or type of calcium supplement and might be used in evaluating the absorptivity of marketed calcium supplements.

Is bone equally responsive to calcium and vitamin D intake from food vs. supplements? Use of 41calcium tracer kinetic model

BackgroundFew interventions directly compare equivalent calcium and vitamin D from dairy vs. supplements on the same bone outcomes. The radioisotope calcium-41 (41Ca) holds promise as a tracer method to directly measure changes in bone resorption with differing dietary interventions. ObjectiveUsing 41Ca tracer methodology, determine if 4servings/day of dairy foods results in greater 41Ca retention than an equivalent amount of calcium and vitamin D from supplements. Secondary objective was to evaluate the time course for the change in 41Ca retention. MethodsIn this crossover trial, postmenopausal women (n=12) were dosed orally with 100nCi of 41Ca and after a 180day equilibration period received dairy (4 servings/day of milk or yogurt; ~1300mg calcium, 400IU cholecalciferol (vitamin D3/day)) or supplement treatments (1200mg calcium carbonate/day and 400IU vitamin D3/day) in random order. Treatments lasted 6weeks separated by a 6week washout (WO). Calcium was extracted from weekly 24h urine collections; accelerator mass spectrometry (AMS) was used to determine the 41/40Ca ratio. Primary outcome was change in 41/40Ca excretion. Secondary outcome was the time course for change in 41Ca excretion during intervention and WO periods. ResultsThe 41/40Ca ratio decreased significantly over time during both treatments; there was no difference between treatments. Both treatments demonstrated a significant retention of 41Ca within 1–2weeks (p=0.0007 and p<0.001 for dairy and supplements, respectively). WO demonstrated a significant decrease (p=0.0024) in 41Ca retention within 1–2weeks, back to pre-intervention levels. ConclusionThese data demonstrate that urinary 41Ca retention is increased with an increase in calcium and vitamin D intake regardless of the source of calcium, and the increased retention occurs within 1–2weeks.

Effect of dairy foods vs. calcium and vitamin‐D supplements on bone metabolism: use of 41Ca tracer (1018.12)

Effect of dairy foods vs calcium &amp; vitamin‐D supplements on bone metabolism: Use of 41Ca tracer E Demmer1, ER Gertz2, T Rogers1, D Hillgonds3, M Garrod2, MD Van Loan2 1. Graduate Group in Nutritional Biology, University of California Davis, Davis, CA 2. USDA/ARS Western Human Nutrition Research Center, Davis, CA 3. Lawrence Livermore National Laboratory, Livermore, CA Calcium(Ca) &amp; vitamin D (D) are essential for bone health but other dietary components may also be important. Dairy products provide a rich supply of Mg, P, K, and protein, all important to bone health. We examined the effect of 4 servings/d milk &amp; yogurt compared to equal intakes of Ca &amp; D from supplements on bone metabolism. Twelve healthy postmenopausal women, who were low‐moderate dairy consumers, completed a randomized crossover trial with two 6 wk interventions separated by a 6 wk washout period. Six months prior to intervention women were dosed with 41Ca to label the skeleton. 24‐hr urine samples were analyzed for 41Ca/40Ca using accelerator mass spectrometry. Both interventions resulted in a significant decline (p &lt; 0.0001) of Ca lost from the skeleton. Reduced resorption for supplement (p=0.0034) and dairy (p= 0.0002) groups was confirmed by CTx. Formation (BAP) significantly increased (p=0.032) in the dairy group only. Ca intake for the dairy group was significantly less (‐120mg/d) than that consumed by supplements, this difference can be attributed to other dietary Ca sources, e.g. increased vegetable and grain intake in the supplement group. Our results suggest that the nutrient‐rich composition of milk and yogurt may be more effective in maintaining bone health in healthy postmenopausal women compared to Ca and D supplements alone.Funding: Dairy Research Institute, California Dairy Council, USDA‐ARS‐Western Human Nutrition Research CenterGrant Funding Source: Dairy Research Institute, California Dairy Council, USDA‐ARS‐Western Human Nutrition Research Center

Calcium metabolism in patients with castration-resistant prostate cancer: Development of a quantitative urinary assay for extent of bone metastasis.

130 Background: In patients with metastatic castration-resistant prostate cancer (CRPC), the degree of perturbation from normal bone turnover provides a strong indication of risk for disease progression, skeletal complications and death. Biomarkers of bone cell function and bone collagen degradation provide an integrated index of the underlying disease for patients with bony metastases, but available bone markers are not precise or accurate. In this study, patients with CRPC with bony metastases will consume a single oral calcium-41 dose and the pharmacokinetics of this will be measured over an 18 month period. Participants will also be assessed clinically for time to progression, skeletal related events and death. Methods: Patients with metastatic castration-resistant prostate cancer and bony metastatses, as diagnosed via bone scintigraphy, who were on bisphosphonates were enrolled into the study. 12 consenting research subjects consumed a single 1.2 microgram 41Ca tracer dose and provided 30-250mL urine specimens (single voids) after dose on day 1 (6h after dosing), days 7, 14, 28, 42, 60, and monthly thereafter. Isotope ratios were measured via accelerator mass spectrometry. Results: Urinary 41Ca/Ca was significantly and inversely associated with increased skeletal tumor burdens suggesting that development of an isotopic urine test for bone metastasis extent is feasible, providing a non-invasive and quantitative measure of disease extent. A calculation of the area under the curve of the measurements between day zero and 14 were inversely correlated with disease extent at baseline. Clinical deterioration with worsening bony disease was associated with a significant decrease in the urinary 41Ca/Ca value. We are currently assessing correlations between bone turnover and outcomes such as therapy effectiveness, disease progression, skeletal related events and death. Conclusions: This work is the first direct measurement of long-term calcium metabolism in advanced prostate cancer, providing a basic scientific complement to cellular and collagen-based measures of bone formation and resorption rates as well as a correlation to clinically relevant outcomes.

Direct measurement of calcium metabolism in patients with castration-resistant prostate cancer using a novel isotope tracing approach in urine.

e15083 Background: Recent cohort studies have shown that in patients with metastatic castration-resistant prostate cancer, the degree of perturbation from normal bone turnover provides a strong indication of risk for disease progression, skeletal complications and death. Biomarkers of bone cell function and bone collagen degradation provide an integrated index of the underlying disease for patients with bony metastases, but available bone markers are insufficiently precise and accurate. In this study, patients with castration-resistant prostate cancer with bony metastases will consume a single oral calcium-41 dose and the pharmacokinetics of this will be measured over an 18 month period. Participants will also be assessed clinically for time to progression, skeletal related events and death. Methods: Patients with metastatic castration-resistant prostate cancer and bony metastatses, as diagnosed via bone scintigraphy, who were on bisphosphonates were enrolled into the study. As part of this ongoing IRB-approved clinical trial, seven consenting research subjects consumed a single 1.2 microgram 41Ca tracer dose and provided 30-250mL urine specimens (single voids) after dose on day 1 (6h after dosing), days 7, 14, 28, 42, 60, and monthly thereafter. Isotope ratios were measured via accelerator mass spectrometry. Results: Urinary 41Ca/Ca was significantly and inversely associated with increasing skeletal tumor burdens suggesting that development of an isotopic urine test for bone metastasis extent is feasible, providing a non-invasive and quantitative measure of disease extent. Data is currently available to 5 months past 41Ca dosing in four of seven subjects, and we are currently assessing correlations between bone turnover and outcomes such as disease progression, skeletal related events and death. Conclusions: This work is the first direct measurement of long-term calcium metabolism in advanced prostate cancer, providing a basic scientific complement to cellular and collagen-based measures of bone formation and resorption rates as well as a correlation to clinically relevant outcomes. Trial accrual is ongoing and additional analyses are expected.

Development of 41Ca-Based Pharmacokinetic Model for the Study of Bone Remodelling in Humans

Initial studies show that 41Ca may be employed as a useful diagnostic bioassay for monitoring metabolic bone disease and its treatment management. The 41Ca-based pharmacokinetic model is developed to assess its feasibility in monitoring bone disease and clinical responsiveness to therapeutic regimens. A four-compartment calcium kinetic model is developed to interpret the results of clinically measured 41Ca tracer kinetics for oral and intravenous dose. This model is extended to simulate changes in bone turnover due to osteoporosis by using Gompertzian function with and without cellular accommodation. The rate constants obtained by fitting to the experimental data on drug intervention are used to simulate the impact of strategic treatment intervention. The present model fits well with the available experimental data on 41Ca tracer kinetics. In the simulated osteoporotic model, the negative bone balance (i.e. bone loss) reflected by 41Ca/Ca urine ratio is used to demonstrate slow/fast increase over time compared to the normal state. The cellular accommodation impact is reflected by a recovery from perturbed balance. The model's predictive ability on the impact of therapeutic intervention is verified using published experimental data. The effect of bisphosphonate intervention results in positive bone balance (i.e. bone gain). The four-compartment 41Ca tracer kinetic model can be flexibly used in the interpretation of results obtained from ongoing clinical studies.

Changes in calcium and vitamin d supplement intake during a one-year falls intervention study

Few interventions directly compare equivalent calcium and vitamin D from dairy vs. supplements on the same bone outcomes. The radioisotope calcium-41 (41Ca) holds promise as a tracer method to directly measure changes in bone resorption with differing dietary interventions.Using 41Ca tracer methodology, determine if 4 servings/day of dairy foods results in greater 41Ca retention than an equivalent amount of calcium and vitamin D from supplements. Secondary objective was to evaluate the time course for the change in 41Ca retention.In this crossover trial, postmenopausal women (n = 12) were dosed orally with 100 nCi of 41Ca and after a 180 day equilibration period received dairy (4 servings/day of milk or yogurt; ~ 1300 mg calcium, 400 IU cholecalciferol (vitamin D3/day)) or supplement treatments (1200 mg calcium carbonate/day and 400 IU vitamin D3/day) in random order. Treatments lasted 6 weeks separated by a 6 week washout (WO). Calcium was extracted from weekly 24 h urine collections; accelerator mass spectrometry (AMS) was used to determine the 41/40Ca ratio. Primary outcome was change in 41/40Ca excretion. Secondary outcome was the time course for change in 41Ca excretion during intervention and WO periods.The 41/40Ca ratio decreased significantly over time during both treatments; there was no difference between treatments. Both treatments demonstrated a significant retention of 41Ca within 1–2 weeks (p = 0.0007 and p < 0.001 for dairy and supplements, respectively). WO demonstrated a significant decrease (p = 0.0024) in 41Ca retention within 1–2 weeks, back to pre-intervention levels.These data demonstrate that urinary 41Ca retention is increased with an increase in calcium and vitamin D intake regardless of the source of calcium, and the increased retention occurs within 1–2 weeks.

A pilot study of the feasibility of long-term human bone balance during perimenopause using a 41Ca tracer

The mechanisms governing calcium fluxes during bone remodeling processes in perimenopausal women are poorly known. Despite higher, albeit erratic, estradiol levels in perimenopause, spine bone loss is greater than during the first five years past the final menstrual flow when estradiol becomes low. Understanding changes during this dynamic transition are important to prevent fragility fractures in midlife and older women. The exploration of long-lived 41Ca (T1/2=1.04×105yrs) tracer measurements using accelerator mass spectrometry (AMS) leads to the possibility of monitoring bone remodeling balance. With this new technology, we explored a pilot long-term feasibility study of bone health by measuring the 41Ca trace element in urine for six years from premenopausal to later perimenopausal phases in one midlife woman. We measured bone mineral density in parallel.

Labeling the human skeleton with 41Ca to assess changes in bone calcium metabolism

Bone research is limited by the methods available for detecting changes in bone metabolism. While dual X-ray absorptiometry is rather insensitive, biochemical markers are subject to significant intra-individual variation. In the study presented here, we evaluated the isotopic labeling of bone using 41Ca, a long-lived radiotracer, as an alternative approach. After successful labeling of the skeleton, changes in the systematics of urinary 41Ca excretion are expected to directly reflect changes in bone Ca metabolism. A minute amount of 41Ca (100 nCi) was administered orally to 22 postmenopausal women. Kinetics of tracer excretion were assessed by monitoring changes in urinary 41Ca/40Ca isotope ratios up to 700 days post-dosing using accelerator mass spectrometry and resonance ionization mass spectrometry. Isotopic labeling of the skeleton was evaluated by two different approaches: (i) urinary 41Ca data were fitted to an established function consisting of an exponential term and a power law term for each individual; (ii) 41Ca data were analyzed by population pharmacokinetic (NONMEM) analysis to identify a compartmental model that describes urinary 41Ca tracer kinetics. A linear three-compartment model with a central compartment and two sequential peripheral compartments was found to best fit the 41Ca data. Fits based on the use of the combined exponential/power law function describing urinary tracer excretion showed substantially higher deviations between predicted and measured values than fits based on the compartmental modeling approach. By establishing the urinary 41Ca excretion pattern using data points up to day 500 and extrapolating these curves up to day 700, it was found that the calculated 41Ca/40Ca isotope ratios in urine were significantly lower than the observed 41Ca/40Ca isotope ratios for both techniques. Compartmental analysis can overcome this limitation. By identifying relative changes in transfer rates between compartments in response to an intervention, inaccuracies in the underlying model cancel out. Changes in tracer distribution between compartments were modeled based on identified kinetic parameters. While changes in bone formation and resorption can, in principle, be assessed by monitoring urinary 41Ca excretion over the first few weeks post-dosing, assessment of an intervention effect is more reliable approximately 150 days post-dosing when excreted tracer originates mainly from bone.

Protocol for assessing bone health in humans by tracing long-lived 41Ca isotope in urine, serum, and saliva samples

2 Slow bone turnover rate and large day-to-day variation in excreted bone biomarkers create a challenge in assessing bone health in humans [1]. The use of the longlived radioisotope 41-calcium (41Ca; t1/2D»10 years), which has a rare natural abundance (41Ca/CaD»10i15), and accelerator mass spectrometry (AMS)1, which is able to detect 41Ca tracer, show great promise in resolving bone health issues [2–4]. However, sample processing for AMS measurements is a barrier to widespread Ca– AMS applications [5]. This communication summarizes an improved protocol for quantifying 41Ca concentrations in biological specimens. Calcium isotope tracers are present in multiple body pools. Saliva sampling is proposed as a noninvasive alternative to blood sampling in a week-long study of two subjects using stable Ca isotopes [6]. Urine is the ultimate noninvasive sample and is known to reXect average plasma levels [4], but high temporal resolution requires direct blood sampling. The previous AMS preparation protocol [4] did not address measurements of either saliva or serum specimens. Our protocol increases sample throughput at a reduced cost and expands routine 41Ca tracer determinations to a wider range of biological specimens including urine, serum, and saliva samples. Urine, fasting blood, and fasting saliva samples were collected at diVerent time points from a healthy post-

Application of 41Ca Tracer and Its AMS Measurement inCIAE

The man-made calcium isotope 41Ca is an ideal tracer for thestudy of calcium metabolism. We represent the first application ofaccelerator mass spectrometry (AMS) measurement of 41Ca tracerin China. The technique is being applied to the research field of cellmessenger at the China Institute of Atomic Energy (CIAE). The samplepreparation methods and the AMS measurements are discussed and someinteresting results are presented.

The study of skeletal calcium metabolism with 41Ca and 45Ca

The living skeleton can be labeled for life by the administration of radiologically trivial amounts of 41Ca tracer. After initial elimination of tracer from the readily exchangeable calcium pools subsequent skeletal calcium turnover maintains and modulates the urine 41Ca content. Uniquely, bone calcium metabolism may then be studied with tracer in near equilibrium with the body's calcium and resorbing calcium directly measured by accelerator mass spectrometry (AMS) of excreta. Our experiments with 25 41Ca labeled subjects demonstrate excellent diurnal stability and remarkable response to intervention of the urine signal. Thus the tracer method may prove a competitive means of measuring the effects of antiresorptive osteoporosis treatments, for therapy development or even clinical monitoring. Novel studies of long-term skeletal evolution are also possible. We realize that routinely administered short-lived calcium radiotracers contain 41Ca impurities and that thousands of experimental participants have been historically inadvertently 41Ca labeled. The 41Ca urine index might now rapidly further be characterized by contemporary measurements of these one-time subjects, and with their by now thoroughly skeleton-equilibrated tracer they might be ideal participants in other new experiments. We are also investigating 45Ca AMS. It may prove preferable to label the skeleton with this radiotracer already familiar to bioscientists, but new to AMS.

Human calcium metabolism including bone resorption measured with 41Ca tracer

Accelerator mass spectrometry is so sensitive to small quantities of 41Ca that it might be used as a tracer in the study of human calcium kinetics to generate unique kinds of data. In contrast with the use of other Ca isotopic tracers, 41Ca tracer can be so administered that the tracer movements between the various body pools achieve a quasi steady state. Resorbing bone may thus be directly measured. We have tested such a protocol against a conventional stable isotope experiment with good agreement.

Biological sample preparation and 41Ca AMS measurement at LLNL

Calcium metabolism in biology may be better understood by the use of 41Ca tracer, although requiring detection by accelerator mass spectrometry (AMS). Methodologies for preparation of urine samples and subsequent AMS measurement were investigated. Novel attempts at preparing CaH 2 were unsuccessful, but CaF 2 of sufficient purity could be produced by precipitation of calcium from urine as oxalate, followed by separation of calcium by cation exchange chromatography and washing the CaF 2 precipitate. The presence of some remaining impurities could be compensated for by selecting the appropriate accelerated ion charge state for AMS. The use of projectile X-rays for isobar discrimination was explored as an alternative to the conventional dE dx detector.