Moderate Zinc Supplementation During Prolonged Steroid Therapy Exacerbates Bone Loss in Rats

The present study was conducted to understand the influence of zinc on bone mineral metabolism in prednisolone-treated rats. Disturbance in bone mineral metabolism was induced in rats by subjecting them to prednisolone treatment for a period of 8 weeks. Female rats aged 6–8 weeks weighing 150 to 200 g were divided into four treatment groups, viz., normal control, prednisolone-treated (40 mg/kg body weight orally, thrice a week), zinc-treated (227 mg/L in drinking water, daily), and combined prednisolone?+?zinc-treated groups. Parameters such as changes in mineral levels in the bone and serum, bone mineral density (BMD), bone mineral content (BMC), and bone 99m-technetium-labeled methylene diphosphonate (^99mTc-MDP) uptake were studied in various treatment groups. Prednisolone treatment caused an appreciable decrease in calcium levels both in the bone and serum and also in bone dry weight, BMC, and BMD in rats. Prednisolone-treated rats when supplemented with zinc showed further reduction in calcium levels, bone dry weight, BMD, and BMC. The study therefore revealed that moderate intake of zinc as a nutritional supplement during steroid therapy could enhance calcium deficiency in the body and accelerate bone loss.     

Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Apport de la scintigraphie au 99mTc-dépréotide pour le diagnostic de lésions osseuses secondaires dans le cancer bronchopulmonaire non à petites cellules stade III–IV

ObjectiveIn non-small cell lung cancer (NSCLC), metastatic bone involvement is usually assessed using conventional ^99mTc-HMDP bone scintigraphy, which has a high sensitivity but a poor specificity. The purpose of this study was to assess the usefulness of the ^99mTc-D scintigraphy for the detection of malignant bone metastases in patients with NSCLC stage III or IV and to compare these results with ^99mTc-HMDP bone scan findings.MethodsNineteen patients (13 M and 6 F, mean age 59 years) with proven NSCLC, suspected to have stage III or IV were enrolled prospectively. All patients underwent whole body ^99mTc-HMDP and ^99mTc-D scintigraphy to detect bone metastases within a mean interval of 14 days. Each focal uptake of ^99mTc-D or ^99mTc-HMDP was considered benign or malignant, leading to positive or negative diagnosis for bone involvement. The final diagnosis of bone metastases was established by a lung specialist, on the basis of additional imaging modalities and of 12 months follow-up.ResultsTwelve bone lesions were identified by ^99mTc-D scintigraphy, 10 were classified as bone metastases and two were classified as inflammatory bone lesions. Four patients were metastatic. Fifty eight bone lesions were detected by ^99mTc-HMDP scintigraphy, 26 of whom were considered malignant, eight patients were thus considered metastatic. Thereby, the two nuclear medicine modalities were concordant for 13 patients, that is 68% of cases and were discordant for six patients, representing 32% of cases. Diagnostic sensitivity, specificity and accuracy of depreotide scintigraphy and ^99mTc-HMDP bone scintigraphy were 75% for both, 93.3 and 73.3%, and 89.5 and 73.3% respectively.ConclusionOur data suggest that depreotide scintigraphy with the same sensitivity, a better accuracy and specificity than those of ^99mTc-HMDP bone scan can detect metastatic bone lesions in patients with NSCLC suspected to have stage III or IV disease.     

Technetium-99m monoclonal antibody fragment (FAb) scintigraphy in the evaluation of small cell lung cancer: a preliminary report

Small cell lung cancer (SCC) has the most rapid growth rate of the four cell types and metastasizes early. Present imaging modalities for staging include chest x-ray, CT, MRI and bone scans. In this preliminary study, we assessed the clinical role of ^99mTc-monoclonal antibody (MOAB) scintigraphy in five patients with histologically proven SCC. Each patient was infused with 20–30 mCi of ^99mTc labeled Fab fragment of MOAB (NR-LU-10, NeoRx, Seattle, Wash.). Total body simultaneous anterior and posterior images were obtained 14–16 h post injection. SPECT images of the chest were obtained through a 360 ° rotation of the gamma camera and recorded on a 62 × 64 × 16 matrix. Images (1.2cm thick) were generated in transaxial, sagittal and coronal views.Fourteen of fifteen chest lesions detected by CT were confirmed by ^99mTc MOAB scintigraphy. Scintigraphy detected one additional chest lesion not seen by CT. Scintigraphy failed to detect a brain lesion (2 cm), a chest lesion, and two adrenal lesions, all of which were seen by CT. In one patient with multiple (more than 10) lesions in the liver, both scintigraphy and CT detected all lesions. Three spine lesions seen on ^99mTc MDP scan and positive for metastasis on MRI concentrated ^99mTc MOAB, but two rib lesions seen on ^99mTc MDP bone scan did not concentrate ^99mTc MOAB. It is concluded from these preliminary results that the potential usefulness of ^99mTc MOAB scintigraphy as a complementary imaging modality in the staging of small cell lung cancer should be investigated further.     

Comparison of 99mTc-3PRGD2 Integrin Receptor Imaging with 99mTc-MDP Bone Scan in Diagnosis of Bone Metastasis in Patients with Lung Cancer: A Multicenter Study

Purpose

^99mTc-3PRGD2, a promising tracer targeting integrin receptor, may serve as a novel tumor-specific agent for single photon emission computed tomography (SPECT). A multi-center study was prospectively designed to evaluate the diagnostic accuracy of ^99mTc-3PRGD2 imaging for bone metastasis in patients with lung cancer in comparison with the conventional ^99mTc-MDP bone scan.

Methods

The patients underwent whole-body scan and chest tomography successively at both 1 h and 4 h after intravenous injection of 11.1 MBq/Kg ^99mTc-3PRGD2. ^99mTc-MDP whole-body bone scan was routinely performed within 1 week for comparison. Three experienced nuclear medicine physicians blindly read the ^99mTc-3PRGD2 and ^99mTc-MDP images. The final diagnosis was established based on the comprehensive assessment of all available data.

Results

A total of 44 patients (29 male, 59±10 years old) with suspected lung cancer were recruited from 4 centers. Eighty-nine bone lesions in 18 patients were diagnosed as metastases and 23 bone lesions in 9 patients were benign. In a lesion-based analysis, ^99mTc-3PRGD2 imaging demonstrated a sensitivity, specificity, and accuracy of 92.1%, 91.3%, and 92.0%, respectively. The corresponding diagnostic values for ^99mTc-MDP bone scan were 87.6%, 60.9%, and 82.1%, respectively in the same patients. ^99mTc-MDP bone scan had better contrast in most lesions, whereas the ^99mTc-3PRGD2 imaging seemed to be more effective to exclude pseudo-positive lesions and detect bone metastases without osteogenesis.

Conclusion

^99mTc-3PRGD2 is a novel tumor-specific agent based on SPECT technology with a promising value in diagnosis of bone metastasis in patients with lung cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01737112","term_id":"NCT01737112"}}NCT01737112     

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca²⁺]_e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca²⁺]_i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca²⁺]_i increased when [Ca²⁺]_e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca²⁺]_e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca²⁺]_e (up to 20 mM [Ca²⁺]_e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca²⁺]_e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca²⁺]_e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca²⁺]_i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca²⁺]_i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C. © 1996 Wiley-Liss, Inc.     

Influence of complex charge and size on the uptake of 99mTc-diphosphonates in osteogenic tissue

The biodistributions of six chromatographically pure ^99mTc-HEDP complexes have been determined in soft tissues, normal bone and osteogenic lesions (induced with a Walker 256 tumor) in Fisher 344 rats. The physical properties of each ^99mTc-HEDP complex including anionic charge, partial molar volume, molecular weight and spectral characteristics are known; thus allowing structure-activity relationships to be drawn. The results indicate that the smallest, low charged, mononuclear ^99mTc-HEDP complexes have the greatest uptake in bone lesions, and the highest lesion to muscle and lesion to normal bone ratios.     

Knee Structural Alteration and BMI: A Cross‐sectional Study

Objective: To describe the associations among BMI, knee cartilage morphology, and bone size in adults. Research Methods and Procedures: A cross‐sectional convenience sample of 372 male and female subjects (mean age, 45 years; range, 26 to 61 years) was studied. Knee articular cartilage defect score (0 to 4) and prevalence (defect score of ≥2), volume, and thickness, as well as bone surface area and/or volume, were determined at the patellar, tibial, and femoral sites using T1‐weighted fat‐saturation magnetic resonance imaging. Height, weight, BMI, and radiographic osteoarthritis were measured by standard protocols. Results: In multivariate analysis in the whole group, BMI was significantly associated with knee cartilage defect scores (β: +0.016/kg/m² to +0.083/kg/m², all p < 0.05) and prevalence (odds ratio: 1.05 to 1.12/kg/m², all p < 0.05 except for the lateral tibiofemoral compartment). In addition, BMI was negatively associated with patellar cartilage thickness only (β = ?0.021 mm/kg/m²; p = 0.039) and was positively associated with tibial bone area (medial: β = +7.1 mm²/kg/m², p = 0.001; lateral: β = +3.2 mm²/kg/m², p = 0.037). Those who were obese also had higher knee cartilage defect severity and prevalence and larger medial tibial bone area but no significant change in cartilage volume or thickness compared with those of normal weight. Discussion: This study suggests that knee cartilage defects and tibial bone enlargement are the main structural changes associated with increasing BMI particularly in women. Preventing these changes may prevent knee osteoarthritis in overweight and obese subjects.     

Autoradiographic localization of technetium-99 methylene diphosphonate in growth sites of young mice

Conflicting reports about the localization of ^99mTc-MDP in bone and cartilage are found in the literature. Possible binding sites include hydroxyapatite and non-osteoid matrix such as immature collagen. The present study used autoradiographs of demineralized and non-demineralized growth sites in young mice to demonstrate localization of ⁹⁹Tc-MDP, and consequently ^99mTc-MDP, in mineralizing cartilage and bone. Uptake of the isotope was seen in mineralizing bone and cartilage, associated with the mineral in contrast to the organic phase. The results indicate that bone seeking radiopharmaceutical uptake (BSRU) may detect alterations in the rate of mineralized phase in growth sites and thus has the potential to disclose skeletal growth disorders.     

Ultrastructural observations on effects of different concentrations of calcium and thyroxine in vitro on larval epidermal cells of Rana catesbeiana tadpoles

Summary During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5mM) calcium (Ca²⁺) in the absence or presence of thyroxine (10⁻⁷ M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca²⁺ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca²⁺ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca²⁺ concentrations (0.5mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation inRana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.     

Capillary electrophoresis of technetium radiopharmaceuticals

Diagnostically used ^99mTc kit radiopharmaceuticals were analyzed using capillary zone electrophoresis with radioactivity detection: ^99mTc-bis(bis(2-ethyloxyethyl)phosphino)ethane (^99mTc-Myoview, ^99mTc-Tetrofosmin), ^99mTc-trans(1,2-bis(dehydro-2,2,5,5,-tetramethyl-3-furanone-4-methylene-amino)ethane)-tris(3-methoxy-1-propyl)phosphine) (^99mTc-Technescan Q12, ^99mTc-Furifosmin), ^99mTc-methoxyisobutylisonitrile (^99mTc-MIBI), ^99mTc- , -ethylenecysteine diethylester dimer (^99mTc-ECD), ^99mTc-d,l-hexamethylene propyleneamine oxime (^99mTc-HMPAO), ^99mTc-diethylenetriaminepentaacetic acid (^99mTc-DTPA), ^99mTc-ethylene hepatobiliary iminodiacetic acid (^99mTc-EHIDA), ^99mTc- , -ethylenecysteine dimer (^99mTc-EC), ^99mTc-mercaptoacetylglycylglycylglycine (^99mTc-MAG₃), ^99mTc-dimercaptosuccinic acid (^99mTc-DMSA), ^99mTc-methylene diphosphonate (^99mTc-MDP) and ^99mNaTcO₄. A pressure-driven capillary zone electrophoresis was employed to detect small anions of high electrophoretic mobility and cations within one run. Effective ^99mTc complex charges could be determined by a neutral internal standard. All complexes showed the expected electrophoretic behaviours in view of their charges. Pure products were obtained for the majority of the studied complexes. In the case of ^99mTc-Q12, ^99mTc-EHIDA and ^99mTc-MDP, complex mixtures were detected. The high potential of CE for the analysis of ^99mTc radiopharmaceuticals could be shown.     

Capillary electrophoresis of 99mtechnetium radiopharmaceuticals

Diagnostically used ^99mTc kit radiopharmaceuticals were analyzed using capillary zone electrophoresis with radioactivity detection: ^99mTc-bis(bis(2-ethyloxyethyl)phosphino)ethane (^99mTc-Myoview, ^99mTc-Tetrofosmin), ^99mTc-trans(1,2-bis(dehydro-2,2,5,5,-tetramethyl-3-furanone-4-methylene-amino)ethane)-tris(3-methoxy-1-propyl)phosphine) (^99mTc-Technescan Q12, ^99mTc-Furifosmin), ^99mTc-methoxyisobutylisonitrile (^99mTc-MIBI), ^99mTc-l,l-ethylenecysteine diethylester dimer (^99mTc-ECD), ^99mTc-d,l-hexamethylene propyleneamine oxime (^99mTc-HMPAO), ^99mTc-diethylenetriaminepentaacetic acid (^99mTc-DTPA), ^99mTc-ethylene hepatobiliary iminodiacetic acid (^99mTc-EHIDA), ^99mTc-l,l-ethylenecysteine dimer (^99mTc-EC), ^99mTc-mercaptoacetylglycylglycylglycine (^99mTc-MAG₃), ^99mTc-dimercaptosuccinic acid (^99mTc-DMSA), ^99mTc-methylene diphosphonate (^99mTc-MDP) and ^99mNaTcO₄. A pressure-driven capillary zone electrophoresis was employed to detect small anions of high electrophoretic mobility and cations within one run. Effective ^99mTc complex charges could be determined by a neutral internal standard. All complexes showed the expected electrophoretic behaviours in view of their charges. Pure products were obtained for the majority of the studied complexes. In the case of ^99mTc-Q12, ^99mTc-EHIDA and ^99mTc-MDP, complex mixtures were detected. The high potential of CE for the analysis of ^99mTc radiopharmaceuticals could be shown.     

Radiopharmacological profiles of tracers under pathophysiological conditions. Bone uptake of 99mTc-phytate in vitamin D deficient rats

The radiopharmaceutical ^99mTc-phytate, formed by reacting the phosphatic ligand, myo-inositol hexakisphosphate with a reduced form of ^99mTcO₄⁻, is widely employed in diagnostic nuclear medicine for scinti-imaging the RE system. Despite it being a compound derived from a ligand containing phosphatic functional moieties it does not concentrate to a significant extent in the osseous tissue following i.v. injection into animals or humans. Neither has there been any clinical report citing its localization in the skeletal tissue consequent to its i.v. injection into patients with different types of pathophysiological conditions/metabolic bone disorders. In the course of our study of the homing characteristics of radiopharmaceuticals into specific organ systems or secondary target sites we found that ^99mTc-phytate could be directed to some extent into the skeletal tissues of experimental rats by altering its physiological milieu—viz. by decreasing blood calcium levels and increasing bone resorption. Such an altered physiologic condition is obtained by experimentally inducing vitamin D deficiency in animals. Our results show that 8–10% of the administered dose could be redirected to the bone matrix but at the expense of the liver. The study also indicates that many so-called organ specific ^99mTc-radiopharmaceuticals could have more than one target tissue/organ system for accumulation consequent to being introduced into the systemic circulation. In a number of instances the secondary target may be masked and may be elicited only under certain specific pathophysiologic conditions.     

Evaluation of 99mTc-mercaptoacetyltripeptides in mice and a baboon

Different derivatives of MAG₃ carrying amino acids such as d- or l-alanine, d-serine, d-2-aminobutyric acid, d-valine or d-phenylglycine were synthesized and their ^99mTc-complexes were evaluated in mice and a baboon. The efficiency of renal handling of the examined ^99mTc-complexes is influenced not only by their lipophilicity but also to a great extent by their configuration and the site of substitution. The renal excretion characteristics of ^99mTc-MAGAG-DA are superior to those of ^99mTc-MAG₃ and the studied ^99mTc-complexes in both animal species.In an attempt to improve the renal handling of ^99mTc-MAG₃ and to evaluate the effect of derivatization we have synthesized different derivatives of MAG₃ in which one or more glycyl groups are replaced by other amino acids such as d- or l-alanine, D-serine, D-2-aminobutyric acid, D-valine or D-phenylglycine. Due to the presence of a chiral centre in the ligand core, exchange labelling of each of the MAG₃ derivatives results in the formation of two diastereomeric technetium complexes. These isomers were separated by HPLC and evaluated in mice. Biodistribution in mice indicates that the efficiency of renal handling of the examined ^99mTc-complexes is not only influenced by their lipophilicity but also to a great extent by their configuration. The renal excretion characteristics of isomer dA of ^99mTc-MAGAG in mice are superior to those of all other studied ^99mTc-complexes and also of the reference compound [¹³¹I]Hippuran. The isomers lB of several alanyl derivatives of ^99mTc-MAG₃ exhibit a pronounced renal retention in both mice and baboon. The results of the evaluation in a baboon confirm the superiority of ^99mTc-MAGAG-dA over ^99mTc-MAG₃ and the other studied ^99mTc-complexes.     

The Value of SPECT/CT in Monitoring Prefabricated Tissue-Engineered Bone and Orthotopic rhBMP-2 Implants for Mandibular Reconstruction

Bone tissue engineering shows good prospects for mandibular reconstruction. In recent studies, prefabricated tissue-engineered bone (PTEB) by recombinant human bone morphogenetic proteins (rhBMPs) applied in vivo has found to be an effective alternative for autologous bone grafts. However, the optimal time to transfer PTEB for mandibular reconstruction is still not elucidated. Thus, here in an animal experiment of rhesus monkey, the suitable transferring time for PTEB to reconstruct mandibular defects was evaluated by ^99mTc-MDP SPECT/CT, and its value in monitoring orthotopic rhBMP-2 implants for mandibular reconstruction was also evaluated. The result of SPECT/CT showed higher ^99mTc-MDP uptake, indicating osteoinductivity, in rhBMP-2 incorporated demineralized freeze-dried bone allograft (DFDBA) and coralline hydroxyapatite (CHA) implants than those without BMP stimulation. ^99mTc-MDP uptake of rhBMP-2 implant peaked at 8 weeks following implantation while CT showed the density of these implants increased after 13 weeks’ prefabrication. Histology confirmed that mandibular defects were repaired successfully with PTEB or orthotopically rhBMP-2 incorporated CHA implants, in accordance with SPECT/CT findings. Collectively, data shows ^99mTc-MDP SPECT/CT is a sensitive and noninvasive tool to monitor osteoinductivity and bone regeneration of PTEB and orthotopic implants. The PTEB achieved peak osteoinductivity and bone density at 8 to 13 weeks following ectopic implantation, which would serve as a recommendable time frame for its transfer to mandibular reconstruction.     

Technetium-99m labeled monoclonal antibodies: Evaluation of reducing agents

We have evaluated five compounds, stannous chloride (SnCl₂), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with ^99mTc. The reduction of ^99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such ^99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no ^99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for ¹²⁵I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with ^99mTc using AA. The AA technique promises to label antibodies with ^99mTc and perhaps with ¹⁸⁶Re, by a simple “kit” procedure.     

A refined method for assessing 99mTc-MDP whole body retention in prostate cancer patients

Whole body retention (WBR) of ^99mTc labeled methylene diphosphonate (MDP) has been shown to significantly differentiate various clinical stages of prostate cancer. Whole body measurements, when performed at 5 min and 24 h after i.v. administration of ^99mTc-MDP, allows for the calculations of percentage whole body retention (% WBR) after one day. The latter can be expressed relative to either the anterior or posterior projection, or in combination as the geometric mean value.In an attempt to better describe the clinical course of prostate cancer patients with bone metastases we have refined the % WBR calculations to include a normalization factor. The latter consists of the mean 24-h value of WBR's as obtained from 10 prostate cancer patients without bony métastases as determined by bone scintigram. These values were determined for each projection to be: anterior = 26.5 ± 4.7%, posterior = 37.5 ± 7.4%, and geometric mean = 31.5 ± 5.7%. The % WBR is then divided by the normalization factor of choice and expressed as (% WBR)^N.These data are used to better express ^99mTc MDP 24-h whole body retentions when following the clinical course of patients with metastatic carcinoma of the prostate. Caution should be exercised when interpreting these data when metabolic bone pathology is present. A false negative (% WBR)^N value will result if an infiltration of the ^99mTc-MDP occurs during administration.     

Nonselective Block by La3+ of Arabidopsis Ion Channels Involved in Signal Transduction

Lanthanide ions such as La³⁺ are frequently used as blockers to test the involvement of calcium channels in plant and animal signal transduction pathways. For example, the large rise in cytoplasmic Ca²⁺ concentration triggered by cold shock in Arabidopsis seedlings is effectively blocked by 10 mm La³⁺ and we show here that the simultaneous large membrane depolarization is similarly blocked. However, a pharmacological tool is only as useful as it is selective and the specificity of La³⁺ for calcium channels was brought into question by our finding that it also blocked a blue light (BL)-induced depolarization that results from anion channel activation and believed not to involve calcium channels. This unexpected inhibitory effect of La³⁺ on the BL-induced depolarization is explained by our finding that 10 mm La³⁺ directly and completely blocked the BL-activated anion channel when applied to excised patches. We have investigated the ability of La³⁺ to block noncalcium channels in Arabidopsis. In addition to the BL-activated anion channel, 10 mm La³⁺ blocked a cation channel and a stretch-activated channel in patches of plasma membrane excised from hypocotyl cells. In root cells, 10 mm La³⁺ inhibited the activity of an outward-rectifying potassium channel at the whole cell and single-channel level by 47% and 58%, respectively. We conclude that La³⁺ is a nonspecific blocker of multiple ionic conductances in Arabidopsis and may disrupt signal transduction processes independently of any effect on Ca²⁺ channels. Received: 28 July 1997/Revised: 13 November 1997     

Radioisotopic Bone Scintigraphy with the Gamma Camera in the Investigation of Prostatic Cancer

Experience with x-rays, strontium-87m scintigraphy, and technetium-99m polyphosphate scintigraphy in the identification of bone metastases in 201 patients with prostatic cancer is reviewed. About 40% of the patients had demonstrable metastases in bone at the time of first presentation.Comparative studies of 247 x-ray and ^87mSr surveys indicated that x-rays failed to detect metastases in 10% of cases where they were identified by ^87mSr but that the isotopic survey similarly failed to detect radiologically evident deposits in 7% of cases.Similar studies comparing ^99mTc polyphosphate surveys with x-ray scans showed that x-rays missed isotopically detected metastases in 12% of cases, but in only one survey out of 67 did the isotope miss radiologically evident deposits. In a series of 32 patients investigated by both isotopic techniques ^99mTc polyphosphate did not fail to detect any metastases and identified deposits in one patient in whom they were missed by ^87mSr scintigraphy. About 15% of both x-ray and ^87mSr surveys gave equivocal results, but only 3% (2 out of 67) of ^99mTc polyphosphate surveys were equivocal.We concluded that ^99mTc polyphosphate bone scintigraphy with the gamma camera was the most reliable of the techniques used for the identification of bone metastases in patients with carcinoma of the prostate. The results of scintigraphy with ^87mSr suggested that serial surveys may provide early evidence of hormone resistance in prostatic cancer.     

Direct labeling of proteins with 99mTc

The direct labeling of antibodies and antibody fragments to form a highly stable bond between technetium and the sulfide groups of proteins is now well established. To optimize this reaction, the antibody protein must have sufficient reactive sulfides available to accept that technetium metal ions that are formed by the reduction of pertechnetate in the presence of a weak complexing agent. The reactive sulfide groups are provided by first reducing a small fraction of the disulfide bridges in the antibody protein or by starting with Fab′ fragments, which already have reactive sulfide groups. When the antibody protein has been appropriately reduced, and the reactive sulfide groups protected by a metal ion with a lower binding affinity than technetium, such as tin or zinc, very high labeling yields of high-affinity-bonded ^99mTc can be achieved. This can be accomplished without loss of immunoreactivity, measured as either affinity or immunoreactive fraction.Side reactions can produce radiochemical impurities such as low-affinity, bound ^99mTc; ^99mTc colloids; ^99mTc peptides or antibody aggregates; or ^99mTc-complexes. Also, pertechnetate ions may be an impurity if the sodium pertechnetate solution added to the reduced antibodies is not completely reduced. The specifics of minimizing these side reactions have not been extensively discussed in the prior literature; however, it is clear that appropriate reduction of the protein prior to labeling and complete removal of the reducing agent, particularly if it contains reactive sulfide groups or is toxic, are critical.One- or two-step ^99mTc-labeling kits for preparing ^99mTc-labeled antibody or antibody fragments are rapidly being introduced for use in clinical nuclear medicine studies. These direct labeling methods employ a common sequence of chemical reactions, although the reducing agents for both the antibody and the [^99mTc]pertechnetate may vary. Different ^99mTc transfer agents may be used, but all transfer agents have the common feature of quickly forming weak to moderately strong complexes with reduced technetium. Most use Sn(II) to reduce the pertechnetate, although other reducing agents can be used.