Darren J. Hillegonds

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†‡

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.

The Bisphosphonate Olpadronate Inhibits Skeletal Prostate Cancer Progression in a Green Fluorescent Protein Nude Mouse Model

Purpose: Metastatic bone disease is one of the major causes of morbidity and mortality in prostate cancer patients. Bisphosphonates are currently used to inhibit bone resorption and reduce tumor-induced skeletal complications. More effective bisphosphonates would enhance their clinical value. Experimental Design: We tested several bisphosphonates in a green fluorescent protein (GFP)–expressing human prostate cancer nude mouse model. The in vivo effects of four bisphosphonates, including pamidronate, etidronic acid, and olpadronate, on bone tumor burden in mice intratibially inoculated with PC-3-GFP human prostate cancer cells were visualized by whole-body fluorescence imaging and X-ray. Results: The PC-3-GFP cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3-GFP cell growth was monitored by GFP fluorescence and the bone destruction was evaluated by X-ray. We showed that 3,3-dimethylaminopropane-1-hydroxy-1,1-diphosphonic acid (olpadronate) was the most effective bisphosphonate treatment in reducing tumor burden as assessed by GFP imaging and radiography. The GFP tumor area and X-ray score significantly correlated. Reduced tumor growth in the bone was accompanied by reduced serum calcium, parathyroid hormone–related protein, and osteoprotegerin. Conclusions: The serum calcium, parathyroid hormone–related protein, and osteoprotegerin levels were significantly correlated with GFP area and X-ray scores. Treatment with olpadronate reduced tumor growth in the bone measured by GFP and X-ray imaging procedures. Imaging of GFP expression enables monitoring of tumor growth in the bone and the GFP results complement the X-ray assessment of bone disease. The data in this report suggest that olpadronate has potential as an effective inhibitor of the skeletal progression of clinical prostate cancer.

High-Throughput Measurement of 41Ca by Accelerator Mass Spectrometry to Quantitate Small Changes in Individual Human Bone Turnover Rates

Biochemical markers of bone turnover suffer from large analytical and natural fluctuations (20-30%), making small differences in bone resorption impossible to resolve. This limits the clinical utility of such markers for individuals with the skeletal complications associated with many disease states (e.g., metastatic cancer, renal failure, osteoporosis). We are developing the capability to measure small changes (5-10%) in bone turnover rate in vivo by tagging the living skeleton with 41Ca. Among the stable and radioactive calcium isotopes, only 41Ca is useful for direct quantitation of bone turnover because it is extremely rare in nature and radiologically benign (105 years half-life, pure electron capture decay). The ratio of this tracer to total calcium remains quantifiable in body fluids and excreta via accelerator mass spectrometry (AMS) for many years following a single physiological-sized oral or intravenous dose. The highly automated AMS instrumentation and streamlined sample preparation allows a single operator to prepare or run more than 100 samples per day—significantly more than other 41Ca programs worldwide. We intend to exploit these measurements for earlier diagnosis of pathological processes and interactive intervention with therapeutic agents, allowing modulation of these agents to obtain the best individual result for a patient.

Effective Determination of the Long-lived Nuclide 41Ca in Nuclear Reactor Bioshield Concretes: Comparison of Liquid Scintillation Counting and Accelerator Mass Spectrometry

The routine application of liquid scintillation counting to (41)Ca determination has been hindered by the absence of traceable calibration standards of known (41)Ca activity concentrations. The introduction of the new IRMM (41)Ca mass-spectrometric standards with sufficiently high (41)Ca activities for radiometric detection has partly overcome this although accurate measurement of stable Ca concentrations coupled with precise half-life data are still required to correct the certified (41)Ca:(40)Ca ratios to (41)Ca activity concentrations. In this study, (41)Ca efficiency versus quench curves have been produced using the IRMM standard, and their accuracy validated by comparison with theoretical calculations of (41)Ca efficiencies. Further verification of the technique was achieved through the analysis of (41)Ca in a reactor bioshield core that had been previously investigated for other radionuclide variations. Calcium-41 activity concentrations of up to 25 Bq/g were detected. Accelerator mass spectrometry (AMS) measurements of the same suite of samples showed a very good agreement, providing validation of the procedure. Calcium-41 activity concentrations declined exponentially with distance from the core of the nuclear reactor and correlated well with the predicted neutron flux.

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

Background 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. Objective 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. Methods 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. Results 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. Conclusion 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.

Accelerator Mass Spectrometry in Protein Analysis

Tools for protein quantitation have advanced through several phases. Common proteins in readily studied sources, albumin and globulins in plasma, were discovered in the nineteenth century and quantified by gravimetric mass determination of chemical precipitates. Finer fractionations and scales led to quantitation of increasingly less common proteins, but the method has obvious limits. Some proteins, enzymes, are quantified by their activity, measured as the increasing chemical product or a shrinking amount of substrate. No absolute quantitation of enzyme concentration is available without having a well-known standard for each enzyme expressing a particular action. Immunochemical methods successfully quantitate proteins, as long as antibodies are selective for the protein under study. Standard curves are required for quantification, since binding affinities are sensitive to assay conditions. Non-specific interactions further confound quantitation. Electrophoretic and chromatographic separations allowed analysis of even smaller concentrations, with quantitation accomplished through various optical properties, such as absorbance, fluorescence, or chemiluminescence. Not all proteins are responsive to the latter effects, and optical density measurements often have large uncertainties and poor sensitivity.

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.