Biomedical subjects
Publications and source records attributed to R Ventura.
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Erythropoietin (EPO) and soluble transferrin receptor (sTfR) in serum have been proposed as indirect biomarkers for the detection of recombinant human EPO (rhEPO) misuse in sport. The purpose of the present study was to investigate the influence of different levels of physical fitness, sport, different training workload during the sport season, and endurance exercise in the concentrations of these serum biomarkers for their application into mathematical models to indirectly detect rhEPO misuse. Serum EPO and sTfR concentrations were measured in 96 elite athletes of various sports along the sport season, in 21 recreational athletes at baseline (non exercising) conditions and in 129 other recreational athletes before and after long-distance races (10 and 21 km). In elite athletes, hemoglobin concentrations and percentage of reticulocytes were also measured, and indirect detection models applied. In recreational athletes, for EPO and sTfR, significant differences were only observed after the 21-km race. In baseline conditions, no differences were observed between recreational and elite athletes for EPO and sTfR. In elite athletes, individual EPO and sTfR concentrations slightly changed over the sport season, with coefficients of variation (CV) of 26.1 % and 9.0 %, respectively. Hemoglobin and reticulocytes were influenced by sport, but their individual variation over the sport season was not physiologically relevant (CV of 3.7 % and 21.3 %, respectively). When applying mathematical models for detection of rhEPO administration, only one elite athlete obtained an individual model score above the established thresholds. Physical fitness, sport and different training workload during the sport season had no substantial effect on serum EPO and sTfR concentrations, except in recreational athletes after a 21-km race. Variations observed in mathematical models to detect EPO administration were mainly due to fluctuation in hemoglobin concentrations, commonly observed in elite athletes.
Knowledge of the stability of drugs in biological specimens is a critical consideration for the interpretation of analytical results. Identification of proper storage conditions has been a matter of concern for most toxicology laboratories (both clinical and forensic), and the stability of drugs of abuse has been extensively studied. This concern should be extended to other areas of analytical chemistry like antidoping control. In this work, the stability of ephedrine derivatives (ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine), and amphetamine derivatives (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA)) in urine has been studied. Spiked urine samples were prepared for stability testing. Urine samples were quantified by GC/NPD or GC/MS. The homogeneity of each batch of sample was verified before starting the stability study. The stability of analytes was evaluated in sterilized and non-sterilized urine samples at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 24 months for sterile urine samples, and for 6 months for non-sterile samples. For short-term stability testing, analyte concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was also studied in sterile urine for up to three cycles. No significant loss of the analytes under study was observed at any of the investigated conditions. These results show the feasibility of preparing reference materials containing ephedrine and amphetamine derivatives to be used for quality control purposes.
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Serum insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P) have been proposed as indirect biomarkers of rhGH misuse in sports. The purpose of the present study was to investigate concentrations of these biomarkers in athletes at different levels of physical fitness and endurance exercise. Serum total IGF-I and P-III-P were measured in 96 elite athletes of various sports along the training season; in 21 recreational athletes at baseline non-exercising conditions and in another 129 recreational athletes before and after long-distance races (10 and 21 km). No differences were evidenced for IGF-I concentrations, but statistically higher values of serum P-III-P were found in elite athletes compared to recreational ones. Among elite athletes, the specific sport did not affect serum IGF-I. However, P-III-P was statistically higher in the sport performed by the youngest athletes (rhythmic gymnastics), even after correction of the logarithm of the concentration by the reciprocal of age. Over the training season, the within-athlete variabilities of IGF-I and P-III-P in elite athletes were low (22.8 % and 21.7 %, respectively). Recreational athletes taking part in a 21 km competition race showed a significant increase in serum values of IGF-I and P-III-P immediately after the event. Exercise workload and age had a significant effect on serum concentration of P-III-P, while age alone affected IGF-I serum concentrations. Therefore, athlete's reference concentration ranges for doping detection should include subjects from as many different ages and sports as possible.
The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes.
Simultaneous occurrence of narrow and broad QRS complex tachycardias in patients with WPW syndrome usually indicates a macroreentry in an orthodromic atrioventricular reentry-tachycardia using the AV node as antegrade and the accessory pathway as retrograde conduction and vice versa in an antidromic circuit. We report on a 32-year-old woman with WPW syndrome presenting with both a narrow and a broad QRS complex tachycardia using two accessory pathways with different unidirectional conduction properties in combination of an exclusively antegrade conducting AV node. This case report describes conventional mapping techniques and ablation of this unusual entity of a WPW syndrome.
Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.
Soluble transferrin receptor (sTfR) has been proposed as an indirect biomarker of the misuse of recombinant human erythropoietin in sport. An extended validation of four commercially available immunoassays for its measurement in serum is presented. Two ELISA techniques (ELISA1: Orion Diagnostica; ELISA2: R&D Systems), an immunoturbidimetric technique (Turbid: Roche Diagnostics), and a nephelometric technique (Nephel: Dade Behring) were investigated. Intra-laboratory precision better than 3% and correct accuracies were obtained for the Turbid and Nephel techniques using autoanalysers. Slightly worse precision (but always better than 11%) and correct accuracies were also obtained in almost all cases for the two ELISA techniques. Inter-laboratory results showed higher concordances for the ELISA procedures (intraclass correlation coefficients of 0.848 for ELISA1 and 0.973 for ELISA2 which was clearly better). Inter-technique correlations were good for the four techniques with lower dispersions found for the techniques using autoanalysers, i.e. Turbid and Nephel. While Turbid and ELISA1 results (expressed in mg/l) were comparable, results obtained with Nephel were approximately 2.7 times lower. The relationship between those three techniques was maintained when compared with ELISA2, which uses different units (nmol/l). We conclude that ELISA2 and Nephel in our hands were the most suitable techniques in terms of sensitivity, precision and accuracy, and adequacy of the calibration curve for the measurement of sTfR in real serum samples. Discrepancies observed in the results obtained with the different sTfR techniques showed that different reference standards were used and harmonization is recommended in order to obtain comparable results.
The Istituto Superiore di Sanità of Rome, Italy, in cooperation with Institut Municipal d'Investigaciò Mèdica of Barcelona, Spain, set up an external quality control program (HAIRVEQ) to evaluate reliability in hair testing for drug abuse by laboratories from the Italian National Health Service. Samples included in the program were real hair samples from drugs consumers. Prior to sending, hair samples were reduced to powdered form, mixed to ensure homogeneity and tested with GC/MS by four Reference Laboratories. Up to now, four different exercises have been concluded and 23 laboratories participated. Samples containing high and low concentrations of opiates, cocaine and metabolites, low concentrations of MDMA and two blank samples, were included in the intercomparison exercises performed in the first year of HAIRVEQ activities. Results show an insufficient performance of participating laboratories. About 82% of laboratories reported incorrect results on a qualitative basis (false positive and false negative results) for some of the submitted samples. More than one-half of laboratories reported quantitative results (60%). On the basis of the calculated z scores, only between 35 and 55% of results reported should be considered as satisfying. Guidelines have to be provided by Italian authorities for method validation as well as set of recommended cut-off concentrations to orientate laboratories in their quality objectives when developing analytical methodologies as tools to improve reliability and consequently performance of hair analysis.
The feasibility of freeze-dried urine samples containing doping agents to be used in intercomparison exercises and/or as reference materials has been evaluated. Freeze-dried urine samples containing caffeine, ephedrine derivatives (ephedrine, methylephedrine, norephedrine, pseudoephedrine and norpseudoephedrine), amphetamine derivatives (amphetamine, metamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine) and testosterone and epitestosterone glucuronides have been evaluated. For preparation of the samples, blank urines previously subjected to filtration for clarification were fortified with standard solutions of the corresponding compounds and filtered under sterile conditions. Some aliquots of the sterile liquid samples were used for homogeneity testing, others were stored at -80 degree C for reference purposes, and the rest were subjected to lyophilisation. Freeze-dried urine samples were stored at 4-8 degree C and their stability was assessed for a period up to 18 months. Results obtained showed minimal differences (lower than 5%) between lyophilised and non-lyophilised aliquots (stored at -80 degree C) at all time periods except for amphetamine (up to 18%) and norpseudoephedrine (up to 10%). Nevertheless, such differences remained constant over the entire period of study, indicating that the loss of analytes was due to the initial lyophilisation process. The loss of analytes in freeze-dried samples was due to their volatility. Furthermore, an increase in pH by 1 unit was observed following reconstitution of samples prepared from drug-free urine of commercial origin.
Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.
UNLABELLED: Chronic kidney disease (CKD) leads to reduced bone mineral density (BMD) in pre-dialysis and dialysis patients. A few studies have used dual-energy x-ray absorptiometry (DEXA) to assess BMD in pre-dialysis CKD patients and have shown low BMD to be highly prevalent. Until now there have been no studies reporting the histological features of low BMD in pre-dialysis CKD patients. AIM: To determine the prevalence and histological features of low BMD in pre-dialysis CKD patients. METHOD: Pre-dialysis CKD patients (n = 103, 46 females/57 males), median creatinine clearance of 29 (10 - 78) ml/min/ 1.73 m2, were evaluated using biochemical analysis and DEXA. Bone biopsies were obtained from those with low BMD. RESULTS: Fifty (48.5%) out of the 103 patients had low BMD (LBD group) and 53 (51.5%) had normal BMD (NBD group). The risk for low BMD was increased in those patients with alkaline phosphatase levels above 190 U/l and intact-PTH (iPTH) below 70 pg/ml (p < 0.05). Demographic and biochemical parameters from both groups were comparable, except for lower body mass index (BMI) in LBD subjects (p = 0.04). Women who had been post-menopausal for at least 1 year comprised 65% (13/20) and 50% (13/26) of the LBD and NBD groups, respectively (p = NS). In 40 LBD patients, bone histomorphometry revealed adynamic bone disease (ABD, 52.5%), osteomalacia (OM, 42.5%) and mixed bone disease (MBD, 5%). Trabecular bone volume (BV/TV) was lower in ABD and OM patients. A nearly significant association was found between ABD and iPTH < or = 150 pg/ml (p = 0.056), whereas higher values of iPTH were associated with OM. Total alkaline phosphatase < or = 190 U/l was significantly associated with ABD, whereas higher values were associated with OM. No correlation was observed between BV/TV and BMD. CONCLUSION: Low BMD is frequent in pre-dialysis CKD patients, and low turnover bone disease, manifesting as ABD and OM, was the hallmark of this bone loss.
The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4+/-6.0 and 87.3+/-5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 microg/ml (r2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 microg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 microg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 degrees C and in liquid urine samples stored at 4, -20, -40 and -80 degrees C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degrees C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.
Intravenous coronary angiography with synchrotron radiation is a novel and minimally invasive technique for coronary imaging. At the Hamburger Synchrotronstrahlungslabor HASYLAB at DESY, a dedicated angiography system has been developed, which has been shown to provide detailed images of coronary artery segments. For each scan, two monochromatic X-ray images below and above the K-edge of iodine were recorded simultaneously. The two images were subtracted logarithmically to produce a maximal contrast enhancement of the iodine. To date, the procedure has been carried out on 379 outpatients. No complications occurred during or after the angiographic procedure, and hospitalization was not required in any subject. The acceptance by patient is extremely high. Five outside reviewers, blinded as to the clinical data or prior angiographic interpretation, reviewed the images for the presence or absence of 70% or more occlusion of a vessel. They reached a sensitivity of 79% and a specificity of 99%. The study has demonstrated that the synchrotron method has satisfactory sensitivity and very high specificity for severe stenoses. The new method has several advantages over magnetic resonance imaging (MRI), electron beam computed tomography (EBCT), and multi-slice computed tomography (MSCT). Neither vascular calcification (CT) nor the presence of metal stents (MRI) impairs the evaluation of perfusion of segments of the coronary arteries. Furthermore, the spatial resolution is three or four times higher using synchrotron angiography, and problems due to respiratory motion are eliminated.
Perphenazine enanthate has been used in wild animals as a tranquilizer during the period of adaptation to new environments to reduce stress, mortalities and injuries. A gas chromatographic procedure for the quantitative measurement of perphenazine in otter urine has been developed and validated. The method involved an enzymatic hydrolysis with beta-glucuronidase-arylsulfatase from Helix pomatia, followed by a solid-phase extraction with Bond Elut Certify cartridges. The resulting organic phase was evaporated, and the dry extract was derivatised with MSTFA to form the O-TMS derivative. The derivatised extracts were analysed by gas chromatography-mass spectrometry using SIM acquisition mode, measuring three diagnostic ions (m/z 246, 372 and 475). Another phenothiazine derivative, fluphenazine, was used as the internal standard (I.S.). Extraction recoveries for perphenazine and I.S. were 87.6 +/- 8.2% (n=4) and 106.7 +/- 13.4% (n=4), respectively. The calibration curves were linear in the range from 4 to 100 ng/ml (r2=0.99). The limits of detection and quantification were estimated as 1.2 and 3.5 ng/ml, respectively. Precision and accuracy obtained in intra-assay studies were in the ranges of 1.3-8.7 and 1.7-19.5%, respectively, using control samples containing 6, 16 and 60 ng/ml of perphenazine. In inter-assay experiments, precision ranged from 4.3 to 14.9% and accuracy from 3.1 to 11.8%. Examples of the application of the perphenazine quantification method in otter urines after administration of perphenazine enanthate are presented.
Attention-deficit hyperactivity disorder (ADHD) is a highly prevalent childhood psychiatric disorder characterized by impaired attention, excessive motor activity and impulsivity. Converging evidence, suggests a primary role of disturbances in brain dopamine (DA) transmission and a role of genetic factors in its pathology. Inbred provide a well-defined and stable genotype for analysis. C57BL/6 (C57) and DBA/2 (DBA) mice are amongst the most studied inbred strains in the behavioral pharmacology of DA, and they differ in several parameters of the DA system that relate directly to behavioral differences. These strains also exhibit several qualitatively different behavior patterns that rely on separate DA networks (e.g. mesoaccumbens vs. nigrostriatal) and on different modes of inheritance. C57 mice are good learners in most tasks also involving associative learning but are totally unable to learn active avoidance although being very active. Moreover, C57 mice show greater novelty-induced locomotor activity than DBA, which is modulated strongly by DA neurons in the ventral tegmental area (VTA) region. Pharmacological studies also indicate a facilitated mesoaccumbens DA transmission in C57 mice when compared to DBAs. Increased density of D2 autoreceptors located on VTA neurons, and lower D2 postsynaptic receptors in the NAS were observed in DBA relative to C57. Activation of D2 autoreceptors inhibits impluse flow, synthesis, and release rates of DA neurons. As would be predicted from their higher D2 autoreceptor: DBA compared to C57 mice show reduced DA synthesis and release within the mesoaccumbens DA system when challenged with DA direct agonists. However, DBA mice are by fare more susceptible than C57s to stress-induced enhanced mesoaccumbens DA release and in stressful situation, they show sustained active behavioral responses whilst C57 adopt extremely passive responses (behavioral despair). Finally, chronic or repeated stress promote opposite adaptation of VTA DA autoreceptors in the two strains and render the hypoactive DBAs as active as the C57 mice. These results indicate that a complex interaction between genetic and environmental factors controls, mesoaccumbens DA functioning and hyperactive phenotype.
The International Olympic Committee and World AntiDoping Agency restricts the use of beta2-agonists and only the inhaled administration of terbutaline, salbutamol, formoterol and salmeterol is permitted for therapeutic reasons. The aim of this study was to develop a test for the quantitation of terbutaline in urine and evaluate different parameters to distinguish between oral and inhaled administration of the drug. Urine samples were collected from asthmatic and non-asthmatic recreational swimmers who had received repeated doses of oral (3x2.5 mg plus 1x5 mg during 24 h) and inhaled (12x0.5 mg in 24 h with half of it being in the last 4 h) racemic terbutaline, and single oral (5 mg) or single inhaled doses (1 mg). Total terbutaline concentrations (free+conjugated) were determined by enzyme-linked immunosorbent assay. Results showed that after oral administrations urinary terbutaline concentrations were higher than those detected after inhalation. For confirmation purposes, a chiral capillary electrophoretic procedure was established and validated. A solid-phase extraction with Bond-Elut Certify cartridges was undertaken, separation performed using a 50 mM phosphate buffer (pH 2.5) containing 10 mM of (2-hydroxypropyl)-beta-cyclodextrin as running buffer and diode-array UV detection set at 204 nm. The proposed procedure is rapid, selective and sensitive allowing quantitation of free terbutaline enantiomers in urine. No statistical differences were found between total free terbutaline concentrations [S-(+)+R-(-)] in urine collected after oral and inhaled administrations of the drug. After oral doses enantiomeric [S-(+)]/[R-(-)] ratios lower than those obtained after inhalation were observed probably due to an enantioselective metabolism that take place in the intestine, but differences between both routes of administration were not statistically significant. Although different trends were observed after oral and inhaled doses in total terbutaline, total free terbutaline concentrations and in ratios between its enantiomers, differences observed were not sufficiently significant to establish cut-off values to clearly distinguish between both routes of administration.