Long-term precision of 18F-fluoride PET skeletal kinetic studies in the assessment of bone metabolism.

(18)F-Fluoride PET allows noninvasive evaluation of regional bone metabolism and has the potential to become a useful tool for assessing patients with metabolic bone disease and evaluating novel drugs being developed for these diseases. The main PET parameter of interest, termed K(i), reflects regional bone metabolism. The aim of this study was to compare the long-term precision of (18)F-fluoride PET with that of biochemical markers of bone turnover assessed over 6 mo. METHODS: Sixteen postmenopausal women with osteoporosis or significant osteopenia and a mean age of 64 y underwent (18)F-fluoride PET of the lumbar spine and measurements of biochemical markers of bone formation (bone-specific alkaline phosphatase and osteocalcin) and bone resorption (urinary deoxypyridinoline) at baseline and 6 mo later. Four different methods for analyzing the (18)F-fluoride PET data were compared: a 4k 3-compartmental model using nonlinear regression analysis (K(i-4k)), a 3k 3-compartmental model using nonlinear regression analysis (K(i-3k)), Patlak analysis (K(i-PAT)), and standardized uptake values. RESULTS: With the exception of a small but significant decrease in K(i-3k) at 6 mo, there were no significant differences between the baseline and 6-mo values for the PET parameters or biochemical markers. The long-term precision, expressed as the coefficient of variation (with 95% confidence interval in parentheses), was 12.2% (9%-19%), 13.8% (10%-22%), 14.4% (11%-22%), and 26.6% (19%-40%) for K(i-3k), K(i-PAT), mean standardized uptake value, and K(i-4k), respectively. For comparison, the precision of the biochemical markers was 10% (7%-15%), 18% (13%-27%), and 14% (10%-21%) for bone-specific alkaline phosphatase, osteocalcin, and urinary deoxypyridinoline, respectively. Intraclass correlation between the baseline and 6-mo values ranged from 0.44 for K(i-4k) to 0.85 for K(i-3k). No significant correlation was found between the repeated mean standardized uptake value measurements. CONCLUSION: The precision and intraclass correlation observed for K(i-3k) and K(i-PAT) was equivalent to that observed for biochemical markers. This study provided initial data on the long-term precision of (18)F-fluoride PET measured at the lumbar spine, which will aid in the accurate interpretation of changes in regional bone metabolism in response to treatment.     

Conventional measurements of GFR using <Superscript>51</Superscript>Cr-EDTA overestimate true renal clearance by 10 percent

It is widely believed that measurement of the area under the plasma clearance curve (AUC) following a single intravenous injection of chromium-51 labelled ethylene diamine tetra-acetic acid ((51)Cr-EDTA) is a gold standard method for determining glomerular filtration rate (GFR). However, there are reports that (51)Cr-EDTA may have a significant extrarenal clearance. The aim of this study was to identify the non-renal component of (51)Cr-EDTA plasma clearance contributing to the AUC measurement of GFR. Seventy healthy postmenopausal women (mean age 60 years, range 45-79 years) were injected with 3 MBq (51)Cr-EDTA and 0.25 MBq iodine-125 labelled human serum albumin and 11 blood samples taken between 0 and 4 h through an indwelling venous cannula. For the first 21 subjects, two complete urine collections were made 0-2 h and 2-4 h after injection, and for the final 49 patients, four 1-h urine collections were made. The mean (51)Cr-EDTA total plasma clearance was 84 ml/min (range 50-132 ml/min). The mean ratio (SEM) of urine to total clearance determined from the cumulative 1-, 2-, 3- and 4-h data was 0.903 (0.018), 0.891 (0.013), 0.898 (0.011) and 0.899 (0.010) respectively and remained constant despite the mean urine concentration decreasing from 122% to 15%/litre during this period. A least squares fit to data from the 238 individual urine collections was used to determine the fraction of the total plasma clearance attributable to renal clearance, alpha(0), and the residual urine volume, delta V. The results were alpha(0)=0.910 (95% CI: 0.889-0.932) and delta V=14 ml (95% CI: -4 to +34 ml). The overestimation of the true renal clearance of (51)Cr-EDTA by the AUC method is believed to be due to the failure of the plasma clearance curve to reach the true terminal exponential by 2 h after injection as usually assumed. As a result, conventional measurements of GFR using (51)Cr-EDTA overestimate the true renal clearance of tracer by approximately 10%.     

Quantitative studies of bone using (18)F-fluoride and (99m)Tc-methylene diphosphonate: evaluation of renal and whole-blood kinetics

We report a study of the renal and whole-blood kinetics of (18)F-fluoride and (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) and their effect on the evaluation of the skeletal kinetics of the two bone tracers. Data were obtained during an investigation of postmenopausal women taking hormone replacement therapy who were compared with untreated, age-matched controls. After intravenous injection of 18F-fluoride (1 MBq), (99m)Tc-MDP (1 MBq), (51)Cr-ethylenediaminetetraacetic acid (51Cr-EDTA) (3 MBq) and (125)I-human serum albumin ((125)I-HSA) (0.25 MBq), multiple blood samples and urine collections were taken between 0 and 4 h after injection. (51)Cr-EDTA data were used to evaluate the glomerular filtration rate (GFR) and the completeness of each timed urine collection. (125)I-HSA data were used to evaluate the plasma volume and the red cell uptake of the other three tracers. At 4 h, the cumulative urine excretions (and standard deviations, SDs) were: (99m)Tc-MDP, 58.2% (4.8%); (18)F-fluoride, 36.1% (5.7%); (51)Cr-EDTA, 81.5% (4.5%). Plots of the renal clearance of (18)F-fluoride against urine volume showed that urine flow rates greater than 5 ml.min-1 were necessary to ensure a constant renal clearance of (18)F and hence stable conditions for the evaluation of bone tracer kinetics. In contrast, a low urine flow rate had no effect on the renal kinetics of (99m)Tc-MDP. For MDP, the evaluation of skeletal kinetics requires data on protein binding so that calculations can be performed for free MDP. In the present study, protein binding of MDP was evaluated from the ratio of total (99m)Tc-MDP renal clearance to GFR based on the principle that free (99m)Tc-MDP is a GFR tracer. Between 0 and 4 h after injection, the fractional protein binding of MDP increased linearly with time, changing from 21+/-5% immediately after injection to 58+/-5% at 4 h. Although red cell uptake of (99m)Tc-MDP was negligible, for (18)F-fluoride around 30% of circulating tracer was transported in red cells. In view of the data showing the rapid transport of (18)F-fluoride across the red cell membrane, bone kinetic data for (18)F are more accurately reported as whole-blood clearance rather than plasma clearance.     

The reliability of cone-beam computed tomography (CBCT) – Generated frontal cephalograms

BackgroundThe purpose of this study was to evaluate the reliability of measurements from cone-beam computed tomography (CBCT)-generated frontal cephalogram.Materials and methodsCBCT and conventional posteroanterior (PA) cephalograms were taken from 30 adult patients. CBCT image was set according to the Frankfurt-Horizontal (FH) plane as the horizontal plane and the midsagittal reference (MSR) plane. The CBCT frontal cephalograms were generated using the orthogonal Raycast method (group CT_raycast), the orthogonal maximum intensity projection (MIP) method (group CT_MIP) after the head reorientation according to the reference planes, and the generator tool provided by the employed 3-dimensional (3D) imaging software (group CT_generator), respectively. The differences between the CBCT-generated frontal cephalograms and conventional PA cephalograms (group PA_ceph) were compared by paired t-test (p < 0.05).ResultsThe significant differences were shown in two measurements for group CT_raycast, in 12 measurements for group CT_MIP, and in eight measurements for group CT_generator. It was confirmed that the CBCT frontal cephalograms, generated by means of the Raycast method (Group CT_raycast), were more comparable to the conventional PA cephalograms in their measurements than were the others (Groups CT_MIP, CT_generator).ConclusionThis study may well suggest that frontal cephalograms derived by 3D CBCT reorientation can be effectively employed in clinical applications.     

Educational and Relational Stressors Associated with Burnout in Korean Medical Students

Objective

This study aimed to examine whether educational stressors and relational stressors are associated with burnout in medical students and to test social support as a moderator between stressors and burnout.

Methods

A total of 263 medical students attending Gyeongsang National University composed the study sample. A standardized questionnaire was used to investigate educational and relational stressors, three dimensions of burnout, and social support of medical students.

Results

The findings showed that overall burnout is very high among Korean medical students, with 9.9% totally burned out. Educational and relational stressors were significantly associated with the risk of burnout in medical students after controlling for socio-demographics and health behaviors. Social support moderated educational and relational stressors on personal accomplishment, but did not moderate stressors on emotional exhaustion and depersonalization.

Conclusion

Burnout level is substantially high among Korean medical students. Educational and relational stressors are significantly associated with burnout risk in Korean medical students. Social support had moderated educational and relational stressors on personal accomplishment. The results suggest that more social support for medical students is needed to buffer stressors on and burnout.     

Korean version of the diagnostic interview for genetic studies: Validity and reliability

The Diagnostic Interview for Genetic Studies (DIGS), developed in 1994 by the National Institute of Mental Health (NIMH), was translated into Korean and tested for reliability and diagnostic validity. Concurrent validity was tested using the Structured Clinical Interview for DSM-IV (SCID) and clinical diagnoses in 53 patients, most of whom had either schizophrenia or bipolar disorder. Inter-rater reliability was tested in 24 patients. Test-retest reliability was also tested in 17 patients. Overall and specific diagnostic validity for the Korean version of DIGS (DIGS-K) was excellent for most diagnoses. Inter-rater and test-retest reliability for overall and specific diagnoses also ranged from fair to excellent. For schizoaffective disorder, the test-retest reliability of DIGS-K was in a fair range, although the level was lower than that of other diagnoses. However, its diagnostic validity and inter-rater reliability was below fair range. In conclusion, DIGS-K appears to be a reliable interview for major psychiatric disorders.     

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

The contraction of cardiac muscle is generated by reciprocal sliding of actin-containing thin filaments and myosin-containing thick filaments in the sarcomere driven by myosin motors. The interaction of the myosin motors with the overlapping thin filament is primarily controlled by calcium-induced structural changes in the thin filament linked to the intracellular calcium transient (1). Calcium ions released in the cytoplasm following an action potential bind to troponin, triggering the movement of tropomyosin around the filament, which uncovers actin sites to which the motors can bind and power contraction (2). However, some of the myosin motors may not be available for actin binding, as they are folded onto the thick filament surface in relaxing conditions (3). Thick filament–based regulatory mechanisms control the release of the myosin motors from the folded conformation and contribute to the regulation of contractility of striated muscle (3–6).Electron microscopy (EM) studies on isolated thick filaments from vertebrate heart muscle showed that the myosin motors in the region of the filament that contains myosin-binding protein C (MyBP-C), the C-zone, are sequestered in helical tracks on the thick filament surface and are folded back onto their tails in an asymmetric conformation called the interacting-heads motif, or IHM (7, 8), originally identified in two-dimensional crystals of dephosphorylated smooth muscle myosin (9). The IHM has also been associated with a biochemical state of myosin with a very low adenosine triphosphate (ATP)-ase rate, called the “super-relaxed” state (10), which is considered to be an OFF state of myosin. A recent X-ray diffraction study of cardiac muscle (11) extended that concept and suggested that in diastole, the resting phase of the cardiac cycle, three distinct motor conformations coexist in the thick filament in roughly equal numbers: folded helical, folded nonhelical, and disordered. The folded helical motors are likely to correspond to the IHM conformation and are confined to the C-zone. All the folded motors would be unavailable for actin binding and therefore OFF, but the disordered motors would constitute a population of constitutively ON motors that are immediately available for actin binding upon activation of the thin filament.Stress sensing in the thick filament can control the release of the myosin motors from the folded states and might be responsible for modulating the strength of contraction of cardiac muscle in response to changes in the afterload (i.e., the arterial pressure) (12). Moreover, the transitions between these motor conformations, together with the calcium-induced structural changes in the thin filament, control the speed of contraction and relaxation (11). According to this mechanosensing paradigm of thick filament regulation, the constitutively ON motors play a fundamental role in the activation of cardiac muscle, as the force generated by these motors immediately after the electrical stimulus triggers a positive mechanosensing feedback loop that controls the number of active motors and the dynamics of contraction. Destabilization of the folded conformations by mutations in myosin and other thick filament proteins can alter the equilibrium between these motor conformations, leading to a hypercontractile phenotype in some hypertrophic cardiomyopathies (HCM) (13, 14). Pharmacological therapies targeting thick filament proteins to treat HCM (15–17) have been aimed at reversing the destabilization of the folded states caused by these mutations.The contractility of the heart is also controlled by phosphorylation of the myosin regulatory light chain (RLC) (18) and by β-adrenergic signaling pathways mediated by phosphorylation of MyBP-C in the thick filament (19) as well as troponin in the thin filament, which are also likely to alter the equilibrium between regulatory conformations of the motors. RLC phosphorylation is essential for the normal function of the heart. The pattern of contraction of the heart may depend on a spatial gradient of RLC phosphorylation across the ventricle wall (20), and a decrease in the level of RLC phosphorylation is associated with heart failure (21, 22). RLC phosphorylation potentiates the contractility of vertebrate and invertebrate striated muscle, an effect that is generally thought to be mediated by disrupting the folded helical conformation of the myosin motors on the thick filament and increasing the number of motors available for interaction with actin during contraction at a given [Ca²⁺] (23, 24). Disordering of the myosin motors on the thick filament by RLC phosphorylation has been shown in in vitro studies on isolated thick filaments (23) and is the main mechanism of thick filament activation in intact striated muscle of tarantula during contraction (25). However, the effect of RLC phosphorylation on the structure of the cardiac thick filament in diastole is unclear. More generally, the large changes in force and the speed of contraction and relaxation of cardiac muscle produced by β-adrenergic agonists are not associated with significant changes in the diastolic structure of the thick filament (26), so they are not simply mediated by increasing the number of ON motors in diastole. Similarly, length-dependent activation (LDA), the cellular correlate of the Frank–Starling law of the heart and a key autoregulatory mechanism that adjusts the cardiac output in response to different extents of diastolic filling (27, 28), seems not to be simply mediated by a stretch-induced change in the structure of the thick filament in diastole (11, 26). Conflicting results have been reported (29), however, and mathematical models have suggested that activation of myosin motors induced by the passive tension transmitted to the thick filament by titin might contribute to LDA in cardiac muscle (30, 31).Here, we investigated the in situ conformation of the myosin motors and its dependence on temperature, RLC phosphorylation, [Ca²⁺], and sarcomere length (SL) in demembranated cardiac trabeculae from rat hearts using the polarized fluorescence from probes on the N- and C-lobes of the RLC (18, 32). We show that, at the low [Ca²⁺] values that maintain the relaxed state and at near-physiological temperature and lattice spacing, the RLC-lobe orientations are consistent with about one-third of the myosin motors being in the folded helical conformation corresponding to the IHM, likely stabilized by MyBP-C in the C-zone of the filament. At the low [Ca²⁺] values that maintain the relaxed state, the folded conformation of the myosin motors is disrupted by cooling but not by RLC phosphorylation or stretch. However, stretching cardiac muscle at higher [Ca²⁺] that partially activates the thin filament triggers a stress-dependent activation of the thick filament and a force increase that is potentiated by RLC phosphorylation. This increase in contractility, induced by RLC phosphorylation and stretch, can be explained by an interfilament signaling pathway that links the stress-dependent activation of the thick filament to the activation state of the thin filament.     

Isorhamnetin-3-O-galactoside Protects against CCl4-Induced Hepatic Injury in Mice

This study was performed to examine the hepatoprotective effect of isorhamnetin-3-O-galactoside, a flavonoid glycoside isolated from Artemisia capillaris Thunberg (Compositae), against carbon tetrachloride (CCl₄)-induced hepatic injury. Mice were treated intraperitoneally with vehicle or isorhamnetin-3-O-galactoside (50, 100, and 200 mg/kg) 30 min before and 2 h after CCl₄ (20 μl/kg) injection. Serum aminotransferase activities and hepatic level of malondialdehyde were significantly higher after CCl₄ treatment, and these increases were attenuated by isorhamnetin-3-O-galactoside. CCl₄ markedly increased serum tumor necrosis factor-α level, which was reduced by isorhamnetin-3-O-galactoside. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase- 2 (COX-2), and heme oxygenase-1 (HO-1) protein and their mRNA expression levels were significantly increased after CCl₄ injection. The levels of HO-1 protein and mRNA expression levels were augmented by isorhamnetin-3-O-galactoside, while isorhamnetin- 3-O-galactoside attenuated the increases in iNOS and COX-2 protein and mRNA expression levels. CCl₄ increased the level of phosphorylated c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38, and isorhamnetin-3-O-galactoside reduced these increases. The nuclear translocation of nuclear factor kappa B (NF-κB), activating protein-1, and nuclear factor erythroid 2-related factor 2 (Nrf2) were signifi cantly increased after CCl₄ administration. Isorhamnetin-3-O-galactoside attenuated the increases of NF_-κB and c-Jun nuclear translocation, while it augmented the nuclear level of Nrf2. These results suggest that isorhamnetin-3-O-galactoside ameliorates CCl₄-induced hepatic damage by enhancing the anti-oxidative defense system and reducing the inflammatory signaling pathways.     

Quantification of [<Superscript>11</Superscript>C]GB67 binding to cardiac α<Subscript>1</Subscript>-adrenoceptors with positron emission tomography: validation in pigs

INTRODUCTION: An increase in human cardiac alpha(1)-adrenoceptor (alpha(1)-AR) density is associated with various diseases such as myocardial ischemia, congestive heart failure, hypertrophic cardiomyopathy and hypertension. Positron emission tomography (PET) with an appropriate radioligand offers the possibility of imaging receptor function in the normal and diseased heart. [(11)C]GB67, an analogue of prazosin, has been shown in rats to have potential as a PET ligand with high selectivity to alpha(1)-AR. However, alpha(1)-AR density is up to ten times higher in rat heart compared to that in man. The aim of the present preclinical study was to extend the previous evaluation to a large mammal heart, where the alpha(1)-AR density is comparable to man, and to validate a method for quantification before PET studies in man. METHODS: Seven [(11)C]GB67 PET studies, with weight-adjusted target dose of either 5.29 MBq kg(-1) (pilot, test-retest and baseline-predose studies) or 8.22 MBq kg(-1) (baseline-displacement studies), were performed in four anaesthetised pigs (39.5 +/- 3.9 kg). Total myocardial volume of distribution (V (T)) was estimated under different pharmacological conditions using compartmental analysis with a radiolabelled metabolite-corrected arterial plasma input function. A maximum possible blocking dose of 0.12 mumol kg(-1) of unlabeled GB67 was given 20 min before [(11)C]GB67 administration in the predose study and 45 min after administration of [(11)C]GB67 in the displacement study. In addition, [(15)O]CO (3,000 MBq) and [(15)O]H(2)O, with weight adjusted target dose of 10.57 MBq kg(-1), were also administered for estimation of blood volume recovery (RC) of the left ventricular cavity and myocardial perfusion (MBF), respectively. RESULTS: [(11)C]GB67 V (T) values (in ml cm(-3)) were estimated to be 24.2 +/- 5.5 (range, 17.3-31.3), 10.1 (predose) and 11.6 (displacement). MBF did not differ within each pig, including between baseline and predose conditions. Predose and displacement studies showed that specific binding of [(11)C]GB67 to myocardial alpha(1)-ARs accounts for approximately 50% of V (T). CONCLUSION: The present study offers a methodology for using [(11)C]GB67 as a radioligand to quantify human myocardial alpha(1)-ARs in clinical PET studies.