Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players

BACKGROUND AND OBJECTIVES: The determination of the carbon isotope ratio in androgen metabolites has been previously shown to be a reliable, direct method to detect testosterone misuse in the context of antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different countries (Argentina, Italy, Japan, South Africa, Switzerland and Uganda) is examined. METHODS: Carbon isotope ratios of selected androgens in urine specimens were determined using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). RESULTS: Urinary steroids in Italian and Swiss populations were found to be enriched in 13C relative to other groups, reflecting higher consumption of C3 plants in these two countries. Importantly, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were found to be well below the established threshold value for positive cases. CONCLUSIONS: The results obtained with the tested diet groups highlight the importance of adapting the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass spectrometry with refined interpretation criteria for positivity and subject-based profiling of steroids

A large sample study of spin relaxation and magnetometric sensitivity of paraffin-coated Cs vapor cells

We have manufactured more than 250 nominally identical paraffin-coated Cs vapor cells (28mm inner diameter bulbs) for multi-channel atomic magnetometer applications. We describe our dedicated cell characterization apparatus. For each cell we have determined the intrinsic longitudinal, Γ 01, and transverse, Γ 02, relaxation rates. Our best cell shows Γ 01/2π≈0.5Hz, and Γ 02/2π≈2Hz. We find a strong correlation of both relaxation rates which we explain in terms of reservoir and spin exchange relaxation. For each cell we have determined the optimal combination of rf and laser powers which yield the highest sensitivity to magnetic field changes. Out of all produced cells, 90% are found to have magnetometric sensitivities in the range of 9to 30fT $\sqrt{\mathrm{Hz}}$ . Noise analysis shows that the magnetometers operated with such cells have a sensitivity close to the fundamental photon shot noise limi

Urinary analysis of four testosterone metabolites and pregnanediol by gas chromatography-combustion-isotope ratio mass spectrometry after oral administrations of testosterone.

The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach

Detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry: implications in postmortem toxicology.

Because GHB (gamma-hydroxybutyrate) is present in both blood and urine of the general population, toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. In this paper, we propose a procedure for the detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Following liquid-liquid and solid-phase extractions, GHB is derivatized to GHB di-TMS before analysis by GC-C-IRMS. Significant differences in the carbon isotopic ratio (delta(13)C-values > 13.5 per thousand) were found between endogenous and synthetic GHB. Indeed, for postmortem blood samples with different GHB concentrations (range: 13.8-86.3 mg/L), we have obtained GHB delta(13)C-values ranging from -20.6 to -24.7 per thousand, whereas delta(13)C-values for the GHB from police seizure were in the range -38.2 to -50.2 per thousand. In contrast to the use of cut-off concentrations for positive postmortem blood GHB concentrations, this method should provide an unambiguous indication of the drug origin

Predicting erythropoietin resistance in hemodialysis patients with type 2 diabetes

<p>Background: Resistance to ESAs (erythropoietin stimulating agents) is highly prevalent in hemodialysis patients with diabetes and associated with an increased mortality. The aim of this study was to identify predictors for ESA resistance and to develop a prediction model for the risk stratification in these patients.</p> <p>Methods: A post-hoc analysis was conducted of the 4D study, including 1015 patients with type 2 diabetes undergoing hemodialysis. Determinants of ESA resistance were identified by univariate logistic regression analyses. Subsequently, multivariate models were performed with stepwise inclusion of significant predictors from clinical parameters, routine laboratory and specific biomarkers.</p> <p>Results: In the model restricted to clinical parameters, male sex, shorter dialysis vintage, lower BMI, history of CHF, use of ACE-inhibitors and a higher heart rate were identified as independent predictors of ESA resistance. In regard to routine laboratory markers, lower albumin, lower iron saturation, higher creatinine and higher potassium levels were independently associated with ESA resistance. With respect to specific biomarkers, higher ADMA and CRP levels as well as lower Osteocalcin levels were predictors of ESA resistance.</p> <p>Conclusions: Easily obtainable clinical parameters and routine laboratory parameters can predict ESA resistance in diabetic hemodialysis patients with good discrimination. Specific biomarkers did not meaningfully further improve the risk prediction of ESA resistance. Routinely assessed data can be used in clinical practice to stratify patients according to the risk of ESA resistance, which may help to assign appropriate treatment strategies.</p&gt

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

BACKGROUND: Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. CONCLUSIONS/SIGNIFICANCE: AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection

A large sample study of spin relaxation and magnetometric sensitivity of paraffin-coated Cs vapor cells

We have manufactured more than 250 nominally identical paraffin-coated Cs vapor cells (30 mm diameter bulbs) for multi-channel atomic magnetometer applications. We describe our dedicated cell characterization apparatus. For each cell we have determined the intrinsic longitudinal, \sGamma{01}, and transverse, \sGamma{02}, relaxation rates. Our best cell shows \sGamma{01}/2\pi\approx 0.5 Hz, and \sGamma{02}/2\pi\approx 2 Hz. We find a strong correlation of both relaxation rates which we explain in terms of reservoir and spin exchange relaxation. For each cell we have determined the optimal combination of rf and laser powers which yield the highest sensitivity to magnetic field changes. Out of all produced cells, 90% are found to have magnetometric sensitivities in the range of 9 to 30 fTHz. Noise analysis shows that the magnetometers operated with such cells have a sensitivity close to the fundamental photon shot noise limit

Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)