Characterization of the 31P nuclear magnetic resonance spectrum from human melanoma tumors implanted in nude mice.

31P nuclear magnetic resonance spectra of human melanoma (BRO) cells implanted in nude mice were obtained both in vitro and in vivo. The tumors were allowed to grow in the right axillary region of six adult Swiss nude mice to a transverse diameter of 1.5-2 cm, at which point the in vivo 31P nuclear magnetic resonance spectra were obtained. The animals were subsequently sacrificed and the tumor perchloric acid extract was studied in vitro. Relative peak areas are comparable in the two experiments with the exception of inorganic phosphate, which is more abundant in vivo than in vitro by a factor of 4. This difference may be attributed to a greater contribution of the necrotic portion of the tumor to the in vivo spectra. Resonance peaks in the spectrum of the extract were identified on the basis of their coincidence with standards added at pH 7 and 10. Non-energy phosphorylated metabolites present in the tumor at high levels include phosphoethanolamine, phosphocholine, glycerol phosphocholine, and uridine-5'-diphospho-N-acetyl glucosamine. Sugar phosphates and 2,3-diphosphoglycerate from blood made minor contributions to the spectrum. The tumor also contained substantial amounts of pyrimidine triphosphates accounting for 34% of the total nucleoside triphosphate pool.

Bone elasticity and ultrasound velocity are affected by subtle changes in the organic matrix.

The mechanical competence of bone can be studied through the measurement of the components of its material elasticity, a property which can vary both in magnitude and in dependence upon orientation (anisotropy). While it is known that the elasticity is largely determined by the mineral constituents of the bone matrix, it is nonetheless clear that it must be also dependent upon the remaining constituents of bone material. In this work, the influence of organic components on the elasticity is explored by altering specific constituents of the bone matrix to varying degrees. This study addresses two questions: first, are the resulting changes in elasticity strongly or weakly dependent upon direction, and second, are they substantially dependent upon the nature and magnitude of the induced matrix alteration? To answer these questions, we performed different chemical manipulations of the bone matrix and measured the changes in elasticity and velocity using the technique of ultrasound critical angle reflectometry. Altering the properties of the organic matrix resulted in substantial and complex changes in the elasticity of bone. The observed changes were strongly dependent upon direction, could not be explained by changes in density alone, and varied strongly with the specific chemical treatment of the matrix. Immersion in urea selectively affected protein components of the organic matrix and resulted in reversible changes in velocity and elasticity, while removal of collagen caused anisotropic decreases and removal of all organic matter caused a collapse of all components of the elasticity. In conclusion, this study confirms that the organic matrix exerts a profound influence on the elasticity and indicates that the measurement of elastic properties at multiple directions is necessary in the assessment of bone mechanical competence.

Reflection ultrasound velocities and histomorphometric and connectivity analyses: correlations and effect of slow-release sodium fluoride.

To better understand how structural and functional bone properties contribute to the changes in bone biomechanical properties revealed by ultrasound critical angle reflectometry (UCR) analysis, we measured both UCR velocities and histomorphometric properties in bone biopsy specimens from 33 osteoporotic patients before and following intermittent slow-release sodium fluoride (SRNaF) and continuous calcium citrate administration. Mean skeletal fluoride exposure was 17 months, and mean skeletal fluoride content was 0.203 +/- 0.088 SD% bone ash. Intermittent SRNaF and continuous calcium citrate promoted significant increases in trabecular thickness (122 +/- 18 SD microm to 131 +/- 20, p = 0.020), mineral apposition rate (0.79 +/- 0.26 to 1.05 +/- 0.40 microm/day, p = 0.014), and a significant decline in eroded surface (3.9 +/- 1.6 to 2.8 +/- 1.4%, p = 0.002). There were also significant increases in node number (0.193 +/- 0.100 to 0.368 +/- 0.245, p < 0.01) and node-to-node strut length (0.076 +/- 0.087 to 0.191 +/- 0.173, p < 0.01) relative to total cancellous area. Cortical UCR velocity did not change but cancellous velocity significantly increased by 97 m/s following therapy (p = 0.0005). When compared against the significant changes in bone histomorphometry and connectivity, the sum of both cancellous and cortical ultrasound velocities was significantly correlated with node number/area (R2 = 0.305, p < 0.0001) and node-to-node strut length/area (R2 = 0.372, p < 0.0001) and to a lesser extent with mineral apposition rate (R2 = 0.106, p = 0.032). Multiple regression analysis demonstrated that 40% of the variance in the sum of the UCR velocities can be accounted for by the variability in these histomorphometric and connectivity parameters. There were no significant correlations between the sum of cortical and cancellous ultrasound velocities and cancellous bone volume (R2 = 0.014, p = 0533), trabecular thickness (R2 = 0.012, p = 0.47), or bone mineral density (R2 = 0.003, p = 0.80). These observations indicate that velocity measurements with the UCR methodology show an improvement in bone elasticity associated, in part, with an improvement in the rate of bone mineralization and an improvement in bone quality at the structural level as shown by microarchitecture.

Assessment by reflection ultrasound method of the effect of intermittent slow-release sodium fluoride-calcium citrate therapy on material strength of bone.

It has been suggested that fluoride therapy, while increasing bone mass, produces bone with inferior mechanical properties. In the present report this hypothesis was tested using a novel reflection ultrasound technique. Transiliac crest bone biopsies were obtained from 16 patients with osteoporosis and vertebral compression fractures (12 women and 4 men, mean age 56 years) before and after approximately 2 years of intermittent slow-release sodium fluoride therapy (25 mg twice a day) combined with continuous calcium citrate supplementation. Samples were analyzed by a reflection ultrasound method, which analyzes ultrasound velocity with a sample site resolution of 200 microns and thus provides a measure of the mechanical property of single trabeculae (material). For the group, mean fractional change in velocity increased 6.1 +/- 2.3% (SEM) from a mean value of 3303 +/- 80 to 3484 +/- 55 m/s (p = 0.028). A total of 13 patients (81%) demonstrated higher velocities after treatment. Thus reflection ultrasound analysis of bone appears to provide a sensitive means of assessing changes in the material property of bone. Furthermore, these results suggest that the treatment regimen utilized in these patients improves strength of bone at the material or trabecular level largely independently of change in bone mass. The combination therapy also increased spinal (L2-L4) bone density for the group as assessed by dual-photon absorptiometry (5.3 +/- 2.0%). There was no significant correlation between the change in ultrasound velocity and bone density (r = 0.0026, p = 0.996).(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride-stimulated [3H]thymidine uptake in a human osteoblastic osteosarcoma cell line is dependent on transforming growth factor beta.

Controversy exists regarding the effect of fluoride on human osteoblast proliferation. To learn more of the cellular action of fluoride, we chose the clonal osteoblast cell line HOS TE85 as a model system. In these phenotypically osteoblast-like cells, sodium fluoride stimulated [3H]thymidine incorporation in a dose-dependent manner over the concentration range 1 x 10(-5)-2 x 10(-4) M. The fluoride-induced stimulation of [3H]thymidine uptake was dependent on cell density, being optimal at subconfluent cell numbers. Stimulation of [3H]thymidine uptake was inhibited by anti-transforming growth factor beta but not by antibody to insulin-like growth factor I or beta 2-microglobulin. Transforming growth factor beta was shown to be a biphasic stimulator of [3H]thymidine uptake in HOS TE85, with maximal stimulation occurring at 0.5 nM transforming growth factor beta. In the presence of fluoride the cells were more sensitive to stimulation by this growth factor, with maximum effect occurring at 0.1 nM. Fluoride did not increase mRNA for transforming growth factor beta following either 8 or 24 h of exposure. We conclude that fluoride activates osteoblast proliferation by modulating the cellular sensitivity to transforming growth factor beta, a known stimulator of bone growth.

Measurement of intrinsic bone quality in vivo by reflection ultrasound: correction of impaired quality with slow-release sodium fluoride and calcium citrate.

The intrinsic (material) quality of cancellous and cortical bone was evaluated in vivo from the measurement of reflection ultrasound velocities in the ulna. In cancellous bone, the reflection ultrasound velocity was inversely correlated with age in normal women (r = -0.48, p = 0.001), with a significantly lower mean value in 32 normal postmenopausal women than in 14 premenopausal women (3124 versus 3341 m/s, p < 0.0001). In 32 untreated osteoporotic women the cancellous bone velocity was lower than in normal postmenopausal subjects (2906 versus 3124 m/s, p = 0.0001). Following treatment with slow-release sodium fluoride plus calcium citrate (mean 2.4 years in 33 osteoporotic patients with no fracture during treatment), the cancellous bone velocity was significantly higher than in untreated osteoporotic women (3082 versus 2906 m/s, p = 0.0002) and was not significantly different from that in normal postmenopausal women. The cortical bone velocity displayed similar trends, but the changes did not attain statistical significance. The measurements were repeated approximately 9 months later in 9 untreated and in 20 treated patients; in 5 additional patients, the measurements were made both before and after 9 months of treatment with slow-release sodium fluoride and calcium citrate. The cancellous bone velocity increased significantly (p = 0.046) in these patients, from 3008 m/s before treatment to 3112 m/s after the first 9 months of treatment. The velocity rose significantly from 3037 to 3167 m/s (p = 0.017) in patients treated for a short time (12-30 months at first measurement), but it did not change in untreated patients or those treated for more than 30 months. Thus, the material quality of cancellous bone decreases with normal aging and is reduced further with the osteoporotic process. This impaired quality may be corrected by treatment with slow-release sodium fluoride plus calcium citrate.

Measurement of mechanical properties of bone material in vitro by ultrasound reflection: methodology and comparison with ultrasound transmission.

An ultrasound reflection technique was designed and implemented to study the mechanical properties of bone material. The technique uses the fact that an ultrasound beam produced in water undergoes total internal reflection off a bone sample at a critical angle formally related to the velocity of a pressure wave in bone. When the plane of scattering is rotated around the normal to the sample surface, the critical angle varies with a periodic dependence dictated by the intrinsic symmetry of the bone structure at the point being examined. Most current measurements of sound velocity are made using transmission techniques. A double-blind intercomparison between this technique and a transmission technique, which was previously validated against tensile mechanical testing, was performed for samples of isotropic materials and of human cortical bone. Strong correlations were found for both sets of samples. For the isotropic materials the velocities were approximately equal, but for bone they were on average 11% higher in reflection than in transmission. This was the result both of the higher frequency employed in reflection (3.5 rather than 2.25 MHz) and of the different effects of sample imperfections on the two measurements. In particular, the reflection technique used in this work studied the surface of the sample, but the ultrasound beam in the transmission method propagated through its interior. In assessing the mechanical properties of bone specimens by ultrasound, the reflection technique samples a discrete bone surface element and the transmission method analyzes the entire volume of the specimen. Thus the reflection technique may yield a measure of the mechanical property of bone trabeculae that is largely unaffected by the mass of the entire specimen, but mass and the structural density of the specimen affect the transmission method.

Glutathione in whole blood: a novel determination using double quantum coherence transfer proton NMR spectroscopy.

Double quantum selective coherence transfer proton NMR spectroscopy has been used to observe glutathione in whole blood. The efficient water suppression of this technique avoids the need to resuspend the cells in D2O, hence avoiding equilibrium and kinetic isotope effects. Using this method we estimate the concentration of glutathione in fresh whole rabbit blood at approximately 1.7 mM.

6-Fluoropyridoxol: a novel probe of cellular pH using 19F NMR spectroscopy.

6-Fluoropyridoxol was evaluated as an intracellular pH indicator. This molecule exhibits exceptional sensitivity to changes in pH (approximately 10 ppm acid/base shift) and a pKa approximately 8.2 appropriate for physiological investigations. Using 19F NMR spectroscopy we determined both intra- and extracellular pH in whole blood and confirmed the measurements using traditional techniques: ion-electrodes and 31P NMR spectroscopy.

A diverging gynecological template for radioactive interstitial/intracavitary implants of the cervix.

The Syed-Neblett perineal template has been in use at our institution since 1984. We have occasionally encountered problems in inserting the needles through the peripheral rows of the template and have seen significant convergence of the needles cephalad to the template. To solve these problems we have modified the Syed-Neblett template. In the new design, the entrance holes are closer together than in the original template, but they are angled so as to achieve a more compact design while maintaining a dose distribution close to that of the Syed-Neblett design. We have seen significantly fewer clinical problems with this design. In addition, a smaller number of needles achieve the same dose distribution; this simplifies their visualization on radiographs.

Regional tumor oximetry: 19F NMR spectroscopy of hexafluorobenzene.

PURPOSE: An accurate method for monitoring oxygen tension (pO2) of individual tumors could be valuable for optimizing treatment plans. We have recently shown that 19F nuclear magnetic resonance (NMR) spin-lattice relaxometry of hexafluorobenzene (HFB) provides a highly sensitive indicator of tumor oxygenation. We have now refined the methodology to provide enhanced precision, and applied the method to investigate dynamic changes in tumor oxygenation. METHODS AND MATERIALS: Dunning prostate adenocarcinoma R3327-AT1 was grown in the form of pedicles on the foreback of male Copenhagen rats. When the tumors reached approximately equal to 1 cm diameter, HFB (20 microl) was administered, either centrally or peripherally, by direct intratumoral (i.T) injection. Local pO2 was determined using pulse-burst saturation recovery (PBSR) 19F NMR spectroscopy on the basis of the spin-lattice relaxation rate, R1. RESULTS: Interrogation of the central region of tumors provided typical values in the range pO2 = 1.4-6.4 mmHg, with a typical stability of +/-2 mmHg over a period of 20 min, when rats breathed 33% O2. Altering the inhaled gas to oxygen or carbogen (95% O2/5% CO2) produced no significant change. In contrast, interrogation of tumor periphery indicated baseline pO2 in the range 7.9-78.9 mmHg. Altering inspired gas produced significant changes (p < 0.0001) with O2 or carbogen, although the change was generally greater with carbogen. In each case, pO2 returned to baseline within 16 min of returning the inhaled gas to baseline. CONCLUSION: We believe this method provides a valuable new approach with the requisite precision and accuracy to investigate tumor pO2.

Non-invasive determination of tumor oxygen tension and local variation with growth.

PURPOSE: The objective was to develop and demonstrate a novel noninvasive technique of measuring regional pO2 in tumors. The method is based on measuring 19F nuclear magnetic resonance spin-lattice relaxation rate (R1 = 1/T1) of perfluorocarbon (PFC) emulsion discretely sequestered in a tumor. METHODS AND MATERIALS: We have examined pO2 in the Dunning prostate tumor R3327-AT1 implanted in a Copenhagen rat. Oxypherol blood substitute emulsion was administered intravenously and became sequestered in tissue. Proton magnetic resonance imaging (MRI) showed tumor anatomy and correlated 19F MRI indicated the distribution of perfluorocarbon. Fluorine-19 spectroscopic relaxometry was used to measure pO2 in the tumor and repeated measurements over a period of 3 weeks showed the variation in local pO2 during tumor growth. RESULTS: Perfluorocarbon initially resided in the vascularized peripheral region of the tumor: 19F nuclear magnetic resonance R1 indicated pO2 approximately 75 torr in a small tumor (approximately 1 cm) in an anesthetized rat. As the tumor grew, the sequestered PFC retained its original distribution. When the tumor had doubled in size the residual PFC was predominantly in the core of the tumor and the pO2 of this region was approximately 1 torr indicating central tumor hypoxia. CONCLUSION: We have demonstrated a novel noninvasive approach to monitoring regional tumor pO2. Given the critical role of oxygen tension in tumor response to therapy this may provide new insight into tumor physiology, the efficacy of various therapeutic approaches, and ultimately provide a clinical technique for assessing individual tumor oxygenation.

Tumor oximetry: demonstration of an enhanced dynamic mapping procedure using fluorine-19 echo planar magnetic resonance imaging in the Dunning prostate R3327-AT1 rat tumor.

PURPOSE: We have developed an enhanced approach to measuring regional oxygen tension (pO(2)) dynamics in tumors. The technique is demonstrated in a group of 8 Dunning prostate rat tumors (R3327-AT1) with respect to respiratory challenge. METHODS AND MATERIALS: Hexafluorobenzene was injected directly into the tumors of anesthetized rats. (19)F nuclear magnetic resonance echo planar imaging relaxometry was performed to obtain maps of regional tumor oxygenation under baseline conditions and when the inhaled gas was changed to oxygen or carbogen. RESULTS: Sequential pO(2) maps required 8 min, with a typical precision of 1-3 torr at 30-100 individual regions across a tumor. When rats breathed 33% oxygen, distinct heterogeneity was observed for baseline oxygenation in each tumor with pO(2) values ranging from hypoxic to greater than 100 torr. Larger tumors showed significantly lower baseline pO(2). Respiratory challenge with oxygen or carbogen produced significant increases in tumor oxygenation with a close correlation between the response to each gas at individual locations. Regions of both small and large tumors responded to respiratory challenge, but the rate was generally much faster in initially well-oxygenated regions. CONCLUSIONS: Regional pO(2) was assessed quantitatively and the response of multiple individual tumor regions observed simultaneously with respect to interventions.

Regional tumor oxygen tension: fluorine echo planar imaging of hexafluorobenzene reveals heterogeneity of dynamics.

PURPOSE: Therapeutic success could be enhanced if therapy were tailored to the characteristics of specific tumors. We have been developing novel approaches to measuring tumor oxygen tension in vivo, and recently reported a method based on 19F nuclear magnetic resonance (NMR) spin lattice echo planar imaging (EPI) relaxometry of hexafluorobenzene (HFB). We have now examined the feasibility of monitoring dynamic changes in regional tumor oxygenation in response to respiratory challenge. Preliminary data in one tumor show distinct differences before and subsequent to irradiation. METHODS AND MATERIALS: Dunning prostate adenocarcinoma R3327-AT1 was grown in the form of pedicles on the foreback of male Copenhagen rats. When the tumors reached approximately 1 cm diameter, HFB (40 microl) was administered by direct intratumoral injection deliberately dispersed to interrogate both central and peripheral regions. Local pO2 was determined using pulse burst saturation recovery 19F NMR EPI on the basis of the spin lattice relaxation rate. RESULTS: Interrogation of both central and peripheral regions of tumors showed bimodal distribution for oxygenation, including many voxels with pO2 < 15 torr. Altering the inspired gas to 100% O2 produced significant elevation for regions with initially high pO2 (P < 0.01), but the temporal course of dynamic changes varied for each voxel. Many voxels with low pO2 showed little response. Following irradiation (20 Gy), tumor oxygenation was significantly elevated and remained high for at least 10 h. CONCLUSION: We believe this method provides a valuable new approach to investigate tumor oximetry that may extend our understanding of tumor physiology, and could have prognostic value.

Regional tumor oxygenation and measurement of dynamic changes.

We recently described a novel approach to measuring regional tumor oxygen tension using (19)F pulse burst saturation recovery (PBSR) nuclear magnetic resonance (NMR) echo planar imaging (EPI) relaxometry of hexafluorobenzene. We now compare oxygen tension measurements in a group of size-matched R3327-AT1 Dunning prostate rat tumors made using this new method with those using a traditional polarographic method: the Eppendorf histograph. Similar oxygen tension distributions were found using the two methods, and both techniques showed that tumors with volume greater than 3.5 cm(3) were significantly (P < 0.0001) less well oxygenated than smaller tumors (volume less than 2 cm(3)). Using the (19)F EPI approach, we also examined the response to respiratory challenge. Increasing the concentration of inspired oxygen from 33% to 100% O(2) produced a significant increase (P < 0.0001) in tumor oxygenation for a group of small tumors. In contrast, no change was observed in the mean pO(2) for a group of large tumors. Consideration of individual tumor regions irrespective of tumor size showed a strong correlation between the maximum pO(2) observed when breathing 100% O(2) compared with mean baseline pO(2). These results further demonstrate the usefulness of (19)F EPI to assess changes in regional tumor oxygenation.

Bone phenotype of the aromatase deficient mouse.

Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (microCT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F- Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment.

Characteristics of the 6-MV photon beam produced by a dual energy linear accelerator.

Clinical dosimetry data are presented for the lower-energy x-ray beam of a Varian Clinac 1800 linear accelerator. This beam has comparable characteristics to single energy linear accelerators with the same stated 6-MV x-ray energy. The nominal beam energy was found to be 5.3 +/- 0.3 MV on the central axis. Beam quality expressed in terms of half-value layer in water was found to vary by less than 10% over the entire field. The surface doses are only slightly, but consistently, larger than those reported in the literature for other 6-MV linacs. Dosimetric results presented include central axis percentage depth dose (% DD) and tissue-maximum ratio (TMR), surface and buildup doses, output factors, and inverse square law applicability. The flatness and symmetry characteristics are within the manufacturer's specifications for both large and small fields.

Properties of the 18-MV photon beam from a dual energy linear accelerator.

The beam characteristics of the 18-MV photon beam of a Varian Clinac 1800 are presented. The clinically relevant parameters of central axis depth dose, tissue-phantom ratios, peak-scatter factors, and relative output factors are discussed and compared to 18-MV beam data previously reported. The nominal beam energy was found to be 18.3 +/- 0.8 MV on the central axis. The beam symmetry and uniformity meet the manufacturer's specifications. The inverse-square law is applicable within 1.4% over the clinically useful range of distances and field sizes. An empirical fit equation for the central axis depth dose is presented.

Connectivity in human cancellous bone by three-dimensional magnetic resonance imaging.

Bone architecture affects strength and resistance to fracture. Trabecular connectivity is now recognized as an important measure of bone quality, and could be useful as an indicator of the osteoporotic condition, as well as a tool for measuring the effectiveness of therapies. We have applied three-dimensional magnetic resonance imaging microscopy to human cancellous bone biopsies, and report the results of connectivity measurements. Sample heterogeneity was examined on the basis of connectivity density for subvolumes. The choice of examination volume had a significant effect on connectivity density measurements, but sample volumes greater than 100 mm3 were found to give stable results. Connectivity density was strongly correlated with nodal density, and two-dimensional estimates of connectivity, but not bone volume fraction. Repeat measurement at constant resolution (69 x 138 x 109 microns, signal-to-noise ratio of about 35) showed reproducibility of about 5% for connectivity density. Our most recent results have significantly enhanced resolution (69 x 69 x 43 microns); bone fraction remained constant, but apparent connectivity density is greater.

Dose distribution around a 3.0-mm type 6702 I-125 seed.

The 3M Company recently produced a special version of the type 6702 seed for use in animal studies of ocular melanoma. The seed consists of a single I-125 impregnated ion exchange resin sphere encapsulated in a 3.0-mm-long titanium cylinder, as opposed to the normal 4.5-mm cylinder containing from three to five resin spheres. Monte Carlo simulations and measurements show that the dose distribution around the 3-mm capsule differs in places by up to 10% from that of the 4.5-mm seed. In addition, the two seed types differ by 12% in the ratio of dose in tissue along the transverse seed axis to apparent activity. These differences must be taken into account when using these seeds.