Correlation between gamma glutamyltransferase fractions and bone quality.

Gamma-glutamyltransferase (GGT) has been recently identified as a bone-resorbing factor. The aim of this study was to investigate the association between plasma GGT fractions levels and bone quality. Plasma GGT fractions were analysed by gel-filtration chromatography. Bone quality was established quantitatively by two micro-CT derived microarchitectural parameters: the BV/TV (mineralised bone volume/total volume), and the SMI (structure model index) that describes the rod-like (low resistant) or plate-like (high-resistant) shape of bone trabeculae. We enrolled 93 patients hospitalised for elective total hip replacement (group Arthrosis, n=46) or for proximal femoral fracture (group Fracture, n=47). Patients within the first quartile of BV/TV (Q1, osteoporotic patients, n=6) showed higher levels of b-GGT fraction [median (min-max): 3.37 (1.42­6.81)] compared to patients with normal bone density (fourth quartile Q4, n=10; 1.40 (0.83­4.36); p=0.0393]. Also, according to SMI, b-GGT value was higher in the subgroup with bone fragility [Q1, n=8: 1.36 (0.43­4.36); Q4, n=8: 5.10 (1.4 ­7.60); p=0.0117]. In conclusion, patients characterised by fragile bone structure showed specifically higher levels of plasma b-GGT activity thus suggesting fractional GGT analysis as a possible biomarker in the diagnosis of osteoporosis.

The role of glucose, insulin and NEFA in regulating tissue triglyceride accumulation: Substrate cooperation in adipose tissue versus substrate competition in skeletal muscle.

BACKGROUND AND AIMS: Metabolic factors initiating adipose tissue expansion and ectopic triglyceride accumulation are not completely understood. We aimed to investigate the independent role of circulating glucose, NEFA and insulin on glucose and NEFA uptake, and lipogenesis in skeletal muscle and subcutaneous adipose tissue (SCAT). METHODS AND RESULTS: Twenty-two pigs were stratified according to four protocols: 1) and 2) low NEFA + high insulin ± high glucose (hyperinsulinaemia-hyperglycaemia or hyperinsulinaemia-euglycaemia), 3) high NEFA + low insulin (fasting), 4) low NEFA + low insulin (nicotinic acid). Positron emission tomography with [18F]fluoro-2-deoxyglucose and [11C]acetate, was combined with [14C]acetate and [U-13C]palmitate enrichment techniques to assess glucose and lipid metabolism. Hyperinsulinaemia increased glucose extraction, whilst hyperglycaemia enhanced glucose uptake in skeletal muscle and SCAT. In SCAT, during hyperglycaemia, elevated glucose uptake was accompanied by greater [U-13C]palmitate-TG enrichment compared to the other groups, and by a 39% increase in de novo lipogenesis (DNL) compared to baseline, consistent with a 70% increment in plasma lipogenic index. Conversely, in skeletal muscle, [U-13C]palmitate-TG enrichment was higher after prolonged fasting. CONCLUSIONS: Our data show the necessary role of hyperglycaemia-hyperinsulinaemia vs euglycaemia-hyperinsulinaemia in promoting expansion of TG stores in SCAT, by the consensual elevation in plasma NEFA and glucose uptake and DNL. In contrast, skeletal muscle NEFA uptake for TG synthesis is primarily driven by circulating NEFA levels. These results suggest that a) prolonged fasting or dietary regimens enhancing lipolysis might promote muscle steatosis, and b) the control of glucose levels, in association with adequate energy balance, might contribute to weight loss.

A New Natural Antioxidant Mixture Protects against Oxidative and DNA Damage in Endothelial Cell Exposed to Low-Dose Irradiation.

Exposure to ionizing radiation during diagnostic procedures increases systemic oxidative stress and predisposes to higher risk of cancer and cardiovascular disease development. Many studies indicated that antioxidants protect against radiation-induced damage and have high efficacy and lack of toxicity in preventing radiation exposure damages. The purpose of this study was to investigate the in vitro protective effect of a new antioxidant mixture, named RiduROS, on oxidative stress generation and DNA double-strand breaks (DSBs) induced by low doses of X-rays in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were treated with RiduROS mixture 24 h before a single exposure to X-rays at an absorbed dose of 0.25 Gy. The production of reactive oxygen species (ROS) was evaluated by fluorescent dye staining and nitric oxide (NO) by the Griess reaction, and DSBs were evaluated as number of γ-H2AX foci. We demonstrated that antioxidant mixture reduced oxidative stress induced by low dose of X-ray irradiation and that RiduROS pretreatment is more effective in protecting against radiation-induced oxidative stress than single antioxidants. Moreover, RiduROS mixture is able to reduce γ-H2AX foci formation after low-dose X-ray exposure. The texted mixture of antioxidants significantly reduced oxidative stress and γ-H2AX foci formation in endothelial cells exposed to low-dose irradiation. These results suggest that RiduROS could have a role as an effective radioprotectant against low-dose damaging effects.

Archaeology. The makers of the Protoaurignacian and implications for Neandertal extinction.

The Protoaurignacian culture is pivotal to the debate about the timing of the arrival of modern humans in western Europe and the demise of Neandertals. However, which group is responsible for this culture remains uncertain. We investigated dental remains associated with the Protoaurignacian. The lower deciduous incisor from Riparo Bombrini is modern human, based on its morphology. The upper deciduous incisor from Grotta di Fumane contains ancient mitochondrial DNA of a modern human type. These teeth are the oldest human remains in an Aurignacian-related archaeological context, confirming that by 41,000 calendar years before the present, modern humans bearing Protoaurignacian culture spread into southern Europe. Because the last Neandertals date to 41,030 to 39,260 calendar years before the present, we suggest that the Protoaurignacian triggered the demise of Neandertals in this area.

Similar patterns of myocardial metabolism and perfusion in patients with type 2 diabetes and heart disease of ischaemic and non-ischaemic origin.

AIMS/HYPOTHESIS: Type 2 diabetes and insulin resistance are often associated with the co-occurrence of coronary atherosclerosis and cardiac dysfunction. The aim of this study was to define the independent relationships between left ventricular dysfunction or ischaemia and patterns of myocardial perfusion and metabolism in type 2 diabetes. METHODS: Twenty-four type 2 diabetic patients--12 with coronary artery disease (CAD) and preserved left ventricular function and 12 with non-ischaemic heart failure (HF)--were enrolled in a cross-sectional study. Positron emission tomography (PET) was used to assess myocardial blood flow (MBF) at rest, after pharmacological stress and under euglycaemic hyperinsulinaemia. Insulin-mediated myocardial glucose disposal was determined with 2-deoxy-2-[(18)F]fluoroglucose PET. RESULTS: There was no difference in myocardial glucose uptake (MGU) between the healthy myocardium of CAD patients and the dysfunctional myocardium of HF patients. MGU was strongly influenced by levels of systemic insulin resistance in both groups (CAD, r = 0.85, p = 0.005; HF, r = 0.77, p = 0.01). In HF patients, there was an inverse association between MGU and the coronary flow reserve (r = -0.434, p = 0.0115). A similar relationship was observed in non-ischaemic segments of CAD patients. Hyperinsulinaemia increased MBF to a similar extent in the non-ischaemic myocardial of CAD and HF patients. CONCLUSIONS/INTERPRETATION: In type 2 diabetes, similar metabolic and perfusion patterns can be detected in the non-ischaemic regions of CAD patients with normal cardiac function and in the dysfunctional non-ischaemic myocardium of HF patients. This suggests that insulin resistance, rather than diagnosis of ischaemia or left ventricular dysfunction, affects the metabolism and perfusion features of patients with type 2 diabetes.

Analysis of image sharpness reproducibility on a novel engineered micro-CT scanner with variable geometry and embedded recalibration software.

This study investigates the reproducibility of the reconstructed image sharpness, after modifications of the geometry setup, for a variable magnification micro-CT (µCT) scanner. All the measurements were performed on a novel engineered µCT scanner for in vivo imaging of small animals (Xalt), which has been recently built at the Institute of Clinical Physiology of the National Research Council (IFC-CNR, Pisa, Italy), in partnership with the University of Pisa. The Xalt scanner is equipped with an integrated software for on-line geometric recalibration, which will be used throughout the experiments. In order to evaluate the losses of image quality due to modifications of the geometry setup, we have made 22 consecutive acquisitions by changing alternatively the system geometry between two different setups (Large FoV - LF, and High Resolution - HR). For each acquisition, the tomographic images have been reconstructed before and after the on-line geometric recalibration. For each reconstruction, the image sharpness was evaluated using two different figures of merit: (i) the percentage contrast on a small bar pattern of fixed frequency (f = 5.5 lp/mm for the LF setup and f = 10 lp/mm for the HR setup) and (ii) the image entropy. We have found that, due to the small-scale mechanical uncertainty (in the order of the voxel size), a recalibration is necessary for each geometric setup after repositioning of the system's components; the resolution losses due to the lack of recalibration are worse for the HR setup (voxel size = 18.4 µm). The integrated on-line recalibration algorithm of the Xalt scanner allowed to perform the recalibration quickly, by restoring the spatial resolution of the system to the reference resolution obtained after the initial (off-line) calibration.

A micro-PET/CT approach using O-(2-[18F]fluoroethyl)-L-tyrosine in an experimental animal model of F98 glioma for BNCT.

The present study focuses on a micro-PET/CT application to be used for experimental Boron Neutron Capture Therapy (BNCT), which integrates, in the same frame, micro-CT derived anatomy and PET radiotracer distribution. Preliminary results have demonstrated that (18)F-fluoroethyl-tyrosine (FET)/PET allows the identification of the extent of cerebral lesions in F98 tumor bearing rat. Neutron autoradiography and α-spectrometry on axial tissues slices confirmed the tumor localization and extraction, after the administration of fructose-boronophenylalanine (BPA). Therefore, FET-PET approach can be used to assess the transport, the net influx, and the accumulation of FET, as an aromatic amino acid analog of BPA, in experimental animal model. Coregistered micro-CT images allowed the accurate morphological localization of the radiotracer distribution and its potential use for experimental BNCT.

Contribution of organ blood flow, intrinsic tissue clearance and glycaemia to the regulation of glucose use in obese and type 2 diabetic rats: a PET study.

BACKGROUND AND AIMS: Chronic hyperglycaemia aggravates obesity and diabetes mellitus. The use of glucose by body organs depends on several factors. We sought to investigate the role of blood flow, intrinsic tissue glucose clearance and blood glucose levels in regulating tissue glucose uptake under fasting conditions (FCs) and in response to acute hyperglycaemia (AH) in obese and type 2 diabetic rats. METHODS AND RESULTS: Thirty-six Zucker rats were studied by positron emission tomography to quantify perfusion and glucose uptake during FC and after AH in the liver, myocardium, skeletal muscle and subcutaneous adipose tissue. Progressively higher glucose uptake rates were observed from lean to obese (p < 0.05) and to diabetic rats (p < 0.05) in all tissues during both FC and AH. In FC, they were increased of 7-18 times in obese rats and 11-30 times in diabetic rats versus controls. Tissue glucose uptake was increased by over 10-fold during AH in controls; this response was severely blunted in diseased groups. AH tended to stimulate organ perfusion in control rats. Tissue glucose uptake was a function of intrinsic clearance and glycaemia (mass action) in healthy animals, but the latter component was lost in diseased animals. Differences in perfusion did not account for those in glucose uptake. CONCLUSIONS: Each organ participates actively in the regulation of its glucose uptake, which is dependent on intrinsic tissue substrate extraction and extrinsic blood glucose delivery, but not on perfusion, and it is potently stimulated by AH. Obese and diabetic rats had an elevated organ glucose uptake but a blunted response to acute glucose intake.

Positron emission tomography and [18F]BPA: a perspective application to assess tumour extraction of boron in BNCT.

Positron emission tomography (PET) has become a key imaging tool in clinical practice and biomedical research to quantify and study biochemical processes in vivo. Physiologically active compounds are tagged with positron emitters (e.g. (18)F, (11)C, (124)I) while maintaining their biological properties, and are administered intravenously in tracer amounts (10(-9)-10(-12)M quantities). The recent physical integration of PET and computed tomography (CT) in hybrid PET/CT scanners allows a combined anatomical and functional imaging: nowadays PET molecular imaging is emerging as powerful pharmacological tool in oncology, neurology and for treatment planning as guidance for radiation therapy. The in vivo pharmacokinetics of boron carrier for BNCT and the quantification of (10)B in living tissue were performed by PET in the late nineties using compartmental models based on PET data. Nowadays PET and PET/CT have been used to address the issue of pharmacokinetic, metabolism and accumulation of BPA in target tissue. The added value of the use of L-[(18)F]FBPA and PET/CT in BNCT is to provide key data on the tumour extraction of (10)B-BPA versus normal tissue and to predict the efficacy of the treatment based on a single-study patient analysis. Due to the complexity of a binary treatment like BNCT, the role of PET/CT is currently to design new criteria for patient enrolment in treatment protocols: the L-[(18)F]BPA/PET methodology could be considered as an important tool in newly designed clinical trials to better estimate the concentration ratio of BPA in the tumour as compared to neighbouring normal tissues. Based on these values for individual patients the decision could be made whether BNCT treatment could be advantageous due to a selective accumulation of BPA in an individual tumour. This approach, applicable in different tumour entities like melanoma, glioblastoma and head and neck malignancies, make this methodology as reliable prognostic and therapeutic indicator for patient undergoing BNCT.

In vitro neutron irradiation of glioma and endothelial cultured cells.

To fully develop its potential boron neutron capture therapy (BNCT) requires the combination of a suitable thermal/epithermal neutron flux together with a selective intake of (10)B-boron nuclei in the target tissue. The latter condition is the most critical to be realized as none of the boron carriers used for experimental or clinical purposes proved at the moment an optimal selectivity for cancer cells compared to normal cells. In addition to complex physical factors, the assessment of the intracellular concentration of boron represent a crucial parameter to predict the dose delivered to the cancer cells during the treatment. Nowadays the dosimetry calculation and then the prediction of the treatment effectiveness are made using Monte Carlo simulations, but some of the model assumption are still uncertain: the radiobiological dose efficacy and the probability of tumour cell survival are crucial parameters that needs a more reliable experimental approach. The aim of this work was to evaluate the differential ability of two cell lines to selectively concentrate the boron-10 administered as di-hydroxyboryl-phenylalanine (BPA)-fructose adduct, and the effect of the differential boron intake on the damage produced by the irradiation with thermal neutrons; the two cell lines were selected to be representative one of normal tissues involved in the active/passive transport of boron carriers, and one of the tumour. Recent in vitro studies demonstrated how BPA is taken by proliferating cells, however the mechanism of BPA uptake and the parameters driving the kinetics of influx and the elimination of BPA are still not clarified. In these preliminary studies we analysed the survival of F98 and human umbilical vein endothelial cells (HUVEC) cells line after irradiation, using different thermal fluencies at the same level of density population and boron concentration in the growing medium prior the irradiation. This is first study performed on endothelium model obtained by a primary human cell line (HUVEC). The perspective application of this work is to develop a model able to foresee the effects produced by different combination of boron influx with a thermal neutron fluencies, applying a standardized radiobiological methodology, and in particular to continue the investigation of the radiobiological effects on the endothelium model as the main tissue involved in the transport of boronated molecules.

Combined TL and 10B-alanine ESR dosimetry for BNCT.

The dosimetric technique described in this paper is based on electron spin resonance (ESR) detectors using an alanine-boric compound acid enriched with (10)B, and beryllium oxide thermoluminescent (TL) detectors; with this combined dosimetry, it is possible to discriminate the doses due to thermal neutrons and gamma radiation in a mixed field. Irradiations were carried out inside the thermal column of a TRIGA MARK II water-pool-type research nuclear reactor, also used for Boron Neutron Capture therapy (BNCT) applications, with thermal neutron fluence from 10(9) to 10(14) nth cm(-2). The ESR dosemeters using the alanine-boron compound indicated ESR signals about 30-fold stronger than those using only alanine. Moreover, a negligible correction for the gamma contribution, measured with TL detectors, almost insensitive to thermal neutrons, was necessary. Therefore, a simultaneous analysis of our TL and ESR detectors allows discrimination between thermal neutron and gamma doses, as required in BNCT.

Colorectal cancer screening programme by faecal occult blood test in Tuscany: first round results.

Screening with faecal occult blood test (FOBT) has been shown to be effective in reducing mortality from colorectal cancer. Tuscany was the first region in Italy in which a screening programme for colorectal cancer by FOBT was initiated region-wide. The aim of the paper was to describe organizational aspects, a quality control model and the results of this experience. From June 2000 to December 2001, 192583 subjects aged 50-70 were invited to undergo a 1-day immunochemical test without any dietary restriction. A total of 78505 subjects (41%) performed the screening test, of whom 4537 responders had a positive test result (5.8%). Among them, 1122 refused any form of assessment or underwent a colonoscopy outside the screening referral centres, with an overall assessment compliance of 75.3%. Malignancies were found in 193 patients and at least a high-risk adenomatous polyp in 692 patients. In about a quarter of the positive subjects who underwent assessment, cancer or high-risk adenoma was detected. In conclusion, data from this experience supported the feasibility of biennial colorectal screening programme by FOBT, particularly regarding invitation compliance and positivity rate. Further efforts are necessary to implement screening extension and to improve data collection.

Determination of benzene at trace levels in air by a novel method based on solid-phase microextraction gas chromatography/mass spectrometry.

A new method for the determination of benzene at trace levels in air is presented. The method consists of the collection of air samples on adsorbent cartridges with simultaneous adsorption of pre-established amounts of D6-labeled internal standard. Desorption from the cartridge is performed by solid-phase microextraction (SPME) with analysis by gas chromatography/mass spectrometry (GC/MS) using an ion trap mass spectrometer. The influence of several parameters (type of SPME fiber, temperature, time, for example) was investigated, and good linearity in the range 10-400 ng of C6D6, with a coefficient of variance (CV) around 3-5%, was obtained. The method was tested by sampling air in a town center in Italy, and a benzene concentration of approximately 50 microg/m(3) was determined. The maximum limit recommended by the European Community is 10 microg/m(3).

Conformational investigation of two isomeric chiral porphyrins: a convergent approach with different techniques.

The conformation in solution of two atropisomeric meso-tetrabinaphthyl porphyrins, used as catalytic precursors in asymmetric synthesis, was studied by means of experimental ((1)H-NMR ROESY, UV-Vis, and circular dichroism) and computational (semiempirical structure optimization, DeVoe's coupled oscillators calculations) methods. UV-Vis and CD spectra are calculated for several molecular models, with a systematic sampling of the conformational space, and compared to the experimental ones, leading to a structural hypothesis which is confirmed by NMR and PM3.

Determination of absolute configuration of acyclic 1,2-diols with Mo2(OAc)4. 1. Snatzke's method revisited.

The method employing dimolybdenum tetraacetate for the assignment of the absolute configuration of optically active 1,2-diols is thoroughly revisited and applied to several compounds, some of which were synthesized by asymmetric cis-dihydroxylation. No exceptions were found to the empirical rule relating the sign of the induced CD spectrum and the configuration of the substrate, whatever its structure and sterical requirements. To broaden the scope of the method, its applicability to critical situations commonly encountered with synthetic products is tested. It is demonstrated that the method can be applied on samples with low chemical and optical purity, and that it may lend itself as a means to estimate the ee. The roles of the water content of the sample and of the diol-to-dimolybdenum ratio are investigated.

[PtCl3(C2H4)]-[AmH]+ complexes containing chiral secondary amines: use as chiral derivatizing agents for the enantiodiscrimination of unsaturated compounds by 195Pt NMR spectroscopy and NMR stereochemical investigation.

Ionic complexes [PtCl3(C2H4)]-[AmH]+, containing chiral secondary amines, constitute a versatile class of chiral derivatizing agents (CDAs) for the enantiomeric purity determination of chiral unsaturated compounds via 195Pt NMR spectroscopy. The NMR conformational analysis allows us to search for the stereochemical basis of their enhanced versatility.

195Pt NMR determination of the enantiomeric purity and absolute configuration of trisubstituted allenes by using [PtCl3(C2H4)]-[(S,S)-(1-NpMeCH)2NH2]+ as CDA.

[figure: see text] The ionic CDA [PtCl3(C2H4)]-[(S,S)-(1-NpMeCH)2NH2]+ produces, on exchange of its coordinated ethylene by chiral trisubstituted allenes, diastereoisomeric mixtures originating distinct 195Pt NMR resonances for the complexed enantiomers, thus allowing the determination of the enantiomeric purity. A reproducible correlation between relative positions of platinum signals due to the complexed enantiomers and their absolute configuration has been found.

Binding properties of human albumin modified by covalent binding of penicillin.

Derivatisation of lysine residues in human albumin was performed in vitro by reaction with penicillin G. This modification reaction has been reported to occur in patients treated with high dosages of the antibiotic. The structure of the modified protein was characterised by mass spectrometry and circular dichroism. The number of the lysine residues involved depends on the time of incubation and on the drug/protein molar ratio. The secondary structure of the modified protein does not change significantly with respect to the native protein. Furthermore, the binding properties of the modified albumin were characterised by CD spectroscopy. Phenylbutazone, diazepam and bilirubin, known to bind to specific binding areas, were used as markers. A decrease of the affinity to the high-affinity binding sites was observed after the modification.

Significance of both negative T waves and stress-induced normalization of the repolarization phase in infarcted patients: a positron-emission-tomography assessment of regulation of myocardial blood flow and viability of myocardium.

BACKGROUND: The clinical correlation of stress-induced normalization of previously negative T waves (NNTW) to regulation of regional myocardial blood flow (MBF) and tissue viability is still being debated. OBJECTIVE: To clarify its meaning. METHODS: We studied 25 patients, who had previously suffered anterior myocardial infarction and for whom negative T waves were recorded on baseline electrocardiographic precordial leads, by means of positron emission tomography. We obtained MBF in the infarcted myocardial regions under resting conditions for all patients, during infusion of dipyridamole (17 patients) and dobutamine (20 patients), using [13N]-ammonia as a flow tracer. RESULTS: During stress tests, 13 patients exhibited NNTW (group 1) whereas the remaining 12 presented persistent negative T waves (group 2). NNTW was observed in 18 stress studies (for 10 and eight patients during administration of dobutamine and dipyridamole, respectively) whereas persistent negative T waves occurred 19 times (for 10 patients during infusion of dobutamine and nine patients during administration of dipyridamole). A complete concordance of the modifications of the repolarization phase was observed for patients who were subjected both to dipyridamole and to dobutamine studies. Furthermore, we assessed viability of myocardium in 20 of 25 patients using [18F]-fluorodeoxyglucose. For the remaining five patients not subjected to metabolic imaging, a coronary reserve of 1.65 was considered a cut-off of viability. Resting MBF for patients in groups 1 and 2 were similar (0.53 +/- 0.20 versus 0.47 +/- 0.17 ml/min per g, respectively, NS) whereas during pharmacological stress, MBF of patients in group 1 was significantly higher than that for patients in group 2 (0.99 +/- 0.41 versus 0.56 +/- 0.26 ml/min per g, respectively, P < 0.0001). Coronary vasodilating capability, expressed as stress/resting MBF ratio, turned out to be 1.88 +/- 0.49 and 1.16 +/- 0.37 for patients in groups 1 and 2, respectively (P < 0.0001). We observed no difference in mean exercise work load (9.6 +/- 2.80 versus 8.46 +/- 2.18 min, NS) and rate- pressure product (24230 +/- 6425 versus 24207 +/- 8146 mmHg beats/ min, NS) at peak for the two categories of patients. All 13 patients in group 1 (100%) had viable myocardium in the anterior infarcted areas whereas only one of 12 patients in group 2 did (9%, P< 0.0001 versus group 1). Finally, a subanalysis for the specific pharmacological agent used was performed and it gave similar results. CONCLUSION: Regardless of the specific stress test able to elicit the electrocardiographic sign, infarcted dysfunctional areas with stress-induced NNTW were demonstrated to have a higher coronary vasodilating capability and a greater probability of viability of myocardium than had persistent negative T wave regions. Therefore, detection of NNTW appears to be a cheap first-line method for the identification both of a better preserved coronary microcirculatory function and of the persistence of viability of myocardium in the infarcted areas.