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Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3–6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration. [ABSTRACT FROM AUTHOR]

The precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls. Extraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to 'total', 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle. The intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on 'total' and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R2 > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and 'total' GH was evaluated, indicating differences between the various donor groups, but only with limited significance. The top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future. • Detection and quantification of intact 22 kDa and 20 kDa isoforms of GH using mass spectrometry • Combined detection of GH isoforms using bottom-up proteomics • Application of test method to serum samples of healthy individuals and patients with acromegaly • Potential application of analytical approach in sports drug testing [ABSTRACT FROM AUTHOR]

According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR 3 -IGF-I, R 3 -IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection. Noteworthy, the usage of a specific antibody for LongR 3 -IGF-I enables the determination in low ng/mL levels despite the presence of an enormous excess of endogenous human IGF-I. In addition, different metabolism studies ( in - vitro and in - vivo ) were performed using sophisticated strategies such as incubation with skin tissue microsomes, degradation in biological fluids (for all analogues), and administration to rats (for LongR 3 -IGF-I). Herewith, several C-and N-terminally truncated metabolites were identified and their relevancy was additionally confirmed by in - vivo experiments with rodents. Especially for LongR 3 -IGF-I, a metabolite ((Des1-11)-LongR 3 -IGF-I) was identified that prolonged the detectability in - vivo by a factor of approximately 2. The method was validated for qualitative interpretation considering the parameters specificity, identification capability, recovery (26–60%), limit of detection (0.5 ng/mL), imprecision (< 25%), linearity, stability, and matrix effects. A stable isotope labelled ( 15 N)-IGF-I was used as internal standard to control all sample preparation steps. [ABSTRACT FROM AUTHOR]

The numbers of vitamin D inadequacies has reportedly increased in the general population, especially in the Northern hemisphere. However, routine measurement of 25(OH) vitamin D is usually associated with a substantial effort due to the requirement of a venous blood sample taken by medical professionals. Thus, the objective of this work is to develop and validate an easy and minimal-invasive method, using a microsampling technique for autonomous blood collections by medically untrained individuals. The assay enables a simplified monitoring of the vitamin D -status in both, risk group and normal population throughout the year. For this purpose, a simple methanol extraction without derivatization combined with a UHPLC-HRMS method was developed to quantify 25(OH)D2 and 25(OH)D3 in capillary blood. For sample collection, a 20 μl Mitra® device with VAMS® technology is used. By employing the six-fold deuterium-labelled 25(OH)D3 as internal standard, the validated assay provides accurate (<10%) and precise (<11%) results. With a LOQ of 5 ng/ml, the approach also proved sensitive enough to adequately identify potential vitamin D deficiencies (< 12 ng/ml), and proof-of-concept analyses of authentic VAMS® samples (n = 20) yielded test results in the expected blood concentration range. Implementing VAMS® sampling for vitamin D -status monitoring enables a higher frequency due to a simplified, straightforward, and time-effective sample collection. VAMS® assures accurate sample volumes because of its absorptive capacities and, thus, area bias and homogeneity issues associated with conventional DBS are avoided. Regular monitoring of 25(OH)D status throughout the year supports people in high-risk groups for vitamin D -deficiency by early identifying inadequacies and, thus, preventing adverse health consequences. • Bioanalytical method for determination of 25OHD 2 and 25OHD 3 using volumetric blood microsampling. • Sensitive quantification of 25OHD without derivatization. • Precise and accurate quantitative results superior to non-volumetric dried blood spots (DBS). • Established approach facilitates regular vitamin D status-monitoring e.g. in risk groups. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudo-exercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo -metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo , including particularly monohydroxylated and hydrogenated species. No phase II-metabolites were found in vitro ; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro , e.g . monohydroxylated and demethylated species. Here, no phase II-metabolites were observed neither using in vitro nor in vivo samples. Based on the generated data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS) represent a sample matrix collected under minimal-invasive, straightforward and robust conditions. DBS specimens have been shown to provide appropriate test material for different analytical disciplines, e.g. , preclinical drug development, therapeutic drug monitoring, forensic toxicology and diagnostic analysis of metabolic disorders in newborns. However, the sample preparation has occasionally been reported as laborious and time consuming. In order to minimize the manual workload and to substantiate the suitability of DBS for high sample-throughput, the automation of sample preparation processes is of paramount interest. In the current study, the development and validation of a fully automated DBS extraction method coupled to online solid-phase extraction using the example of nicotine, its major metabolites nornicotine, cotinine and trans -3′-hydroxycotinine and the tobacco alkaloids anabasine and anatabine is presented, based on the rationale that the use of nicotine-containing products for performance-enhancing purposes has been monitored by the World Anti-Doping Agency (WADA) for several years. Automation-derived DBS sample extracts were directed online to liquid chromatography high resolution/high mass accuracy tandem mass spectrometry, and target analytes were determined with support of four deuterated internal standards. Validation of the method yielded precise (CV <7.5% for intraday and <12.3% for interday measurements) and linear (r 2 > 0.998) results. The limit of detection was established at 5 ng mL −1 for all studied compounds, the extraction recovery ranged from 25 to 44%, and no matrix effects were observed. To exemplify the applicability of the DBS online-SPE LC–MS/MS approach for sports drug testing purposes, the method was applied to authentic DBS samples obtained from smokers, snus users, and e-cigarette users. Statistical evaluation of the obtained results indicated differences in metabolic behavior depending on the route of administration (inhalative versus buccal absorption) in terms of the ratio of nicotine and nornicotine. [ABSTRACT FROM AUTHOR]

Objective Since Goldspink and colleagues identified the expression of the mRNA of an insulin-like growth factor 1 (IGF-1) isoform in response to mechanical stress in 1996, substantial research into the so-called mechano growth factor and its modus operandi followed until today. Promising preclinical results were obtained by using the synthetic, 24-amino acid residues spanning peptide translated from the exons 4–6 of IGF-1Ec (which was later referred to as the mechano growth factor (MGF) peptide), particularly with regard to increased muscle myoblast proliferation. Consequently, the MGF peptide represented a promising drug candidate for the treatment of neuromuscular disorders; however, its misuse potential in sport was also identified shortly thereafter, and the substance (or class of substances) has been considered prohibited according to the regulations of the World Anti-Doping Agency (WADA) since 2005. While various MGF peptide versions have been known to sports drug testing authorities, the occurrence of a ‘full-length MGF’ as offered via illicit channels to athletes or athletes' managers was reported in 2014, arguably being undetectable in doping controls. Methods An aliquot of the product was obtained and the content characterized by state-of-the-art analytical approaches including gel electrophoretic and mass spectrometric (top-down and bottom-up) sequencing approaches. Upon full characterization, its implementation into modified routine doping controls using ultrafiltration, immunoaffinity-based isolation, and nanoliquid chromatography-high resolution/high accuracy mass spectrometry was established. Results A protein with a monoisotopic molecular mass of 12264.9 Da and a sequence closely related to IGF-1Ec (lacking the signal- and propeptide moiety) was identified. The C-terminus was found to be modified by the elimination of the terminal lysine and a R109H substitution. With the knowledge of the compound's composition, existing doping control assays targeting peptide hormones such as IGF-1 and related substances were assessed as to their capability to detect the full-length MGF. The analyte was detectable at concentrations of 0.25 ng/mL using adapted routine test methods employing immunoaffinity purification followed by nanoscale liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Conclusions A potentially performance enhancing ‘full-length’ MGF derivative was identified and successfully implemented into sports drug testing protocols. Future tests are indicated probing for optimized/dedicated detection methods and assessment of efficacy and elimination kinetics of the substance. [ABSTRACT FROM AUTHOR]

Highlights: [•] Whole blood dried on filter paper – a minimal invasive sampling technique. [•] Screening of anabolic steroid esters in dried blood spots for doping control analysis. [•] Direct detection of exogenous testosterone via the intact esters. [•] Enhancement of the assay sensitivity by preparation of the methyloxime derivatives. [•] Liquid chromatography coupled to high sensitivity mass spectrometry. [ABSTRACT FROM AUTHOR]

Abstract: Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be illicitly used in elite sports due to their endurance enhancing effects. Recently, peginesatide, the first representative of a new generation of ESAs, referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the USA under the trade name Omontys® for the treatment of anaemic patients. Lacking sequence homology with EPO, it consists of a pegylated homodimeric peptide of approximately 45kDa, and thus, specific approaches for the determination of peginesatide in blood were developed as conventional detection assays for EPO do not allow for the analysis of the EPO-mimetic peptides. However, as urine specimens are the most frequently provided doping control samples and pharmacokinetic studies conducted in rats and monkeys revealed the excretion of the pegylated peptide into urine, a detection method for peginesatide in urine would be desirable. A mass spectrometric assay in human urine was developed consisting of protein precipitation with acetonitrile followed by proteolytic digestion after the removal of the acetonitrile fraction under reduced pressure. Purification and concentration of the resulting proteotypic target peptide was accomplished by means of solid-phase extraction on strong cation-exchange resin prior to liquid chromatographic–tandem mass spectrometric analysis. Method validation was performed for qualitative purposes and demonstrated specificity, precision, linearity as well as sufficient sensitivity (limit of detection: 0.5ng/ml) while proof-of-concept for the applicability of the assay for the determination of peginesatide in authentic urine samples was obtained by analyzing animal in vivo specimens collected after a single i.v. administration of peginesatide over a period of 4 days. [Copyright &y& Elsevier]

Abstract: Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%. [Copyright &y& Elsevier]

Abstract: With the constantly increasing sensitivity and robustness of liquid chromatography–mass spectrometry-based instruments combined with enhanced reproducibility as well as mass accuracy and resolution, LC–MS(/MS) has become an integral part of sports drug testing programs particularly concerning the detection of peptide hormones. Although several of the relevant peptidic drugs such as insulins (Humalog LisPro, Novolog Aspart, etc.), growth hormone releasing peptides (GHRPs, e.g., GHRP-2, GHRP-6, Hexarelin, etc.), and insulin-like growth factors (e.g., IGF-1, IGF-2, long-R3-IGF-1) are currently analyzed using dedicated top-down analytical procedures, i.e. employing specifically tailored sample preparation procedures followed by targeted LC–MS(/MS) measurements focusing on intact analytes, first approaches towards multi-analyte methods have been established. These allow the determination of the prohibited substances in blood and urine doping control specimens following therapeutic applications. In addition, the use of new complementary devices such as ion mobility analyzers, e.g., in hybrid mass spectrometers yielded promising data for the differentiation of isobaric insulins, which outlines the potential to further accelerate and multiplex doping control analytical assays to meet the continuously increasing demands of rapid and unambiguous test methods. Moreover, the potential of LC–MS/MS to target recombinant peptide hormones such as human growth hormone using bottom-up approaches has been demonstrated by targeting proteotypic peptides that unambiguously differentiate the recombinant molecule from the naturally occurring and endogenously produced analog. [Copyright &y& Elsevier]

Rationale and Objectives: Magnetic resonance imaging olfactory bulb (OB) volumetry (OBV) is already used as a complementary prognostic tool to assess olfactory disorders. However, a reference standard in imaging for OBV has not been established. The aim of this in vitro study was to compare volumetric results of different magnetic resonance sequences for OBV at 3 T to genuine OB volumes measured by water displacement. Materials and Methods: The volumes of 15 human cadaveric OBs were measured using the water displacement method in this institutional review board–approved prospective study. The magnetic resonance imaging protocol at 3 T included constructive interference in steady state (CISS), T2-weighted (T2w) three-dimensional (3D) sampling perfection with application-optimized contrasts using different flip-angle evolutions (SPACE), T2w two-dimensional (2D) turbo spin-echo (TSE), and T1-weighted (T1w) 3D fast low-angle shot (FLASH) sequences. Two blinded observers independently performed two OB volumetric assessments per bulbus and sequence. Intraobserver and interobserver reliabilities were assessed by intraclass correlation coefficients. Bland-Altman plots were analyzed to evaluate systematic biases and concordance correlation coefficients to assess reproducibility. Results: For both observers, intraclass correlation coefficient analysis yielded almost perfect results for intraobserver reliability (CISS, 0.94–0.98; T2w 3D SPACE, 0.93–0.98; T2w 2D TSE, 0.98–0.98; T1w 3D FLASH, 0.95–0.99). Interobserver reliability showed almost perfect agreement for all sequences (CISS, 0.98; T2w 3D SPACE, 0.89; T2w 2D TSE, 0.93; T1w 3D FLASH, 0.97). The CISS sequence yielded the highest mean concordance correlation coefficient (0.95) and the highest combination of precision (0.97) and accuracy (0.98) values. In comparison with the water displacement method, Bland-Altman analyses revealed the lowest systematic bias (−0.5%) for the CISS sequence, followed by T1w 3D FLASH (−1.3%), T2w 3D SPACE (–7.5%), and T2w 2D TSE (−10.9%) sequences. Conclusions: Compared to the water displacement method, the CISS sequence is suited best to validly and reliably measure OB volumes because of its highest values for accuracy and precision and lowest systematic bias. [Copyright &y& Elsevier]

Rationale and Objectives: The aim of this study was to compare different sequences for olfactory bulb volumetry using 3-T magnetic resonance imaging, evaluating reproducibility, repeatability, and systematic biases. Materials and Methods: Twenty-two volunteers underwent 3-T magnetic resonance imaging of the frontal skull base in this prospective study. Imaging included constructive interference in steady state (CISS), T2-weighted (T2w) three-dimensional (3D) sampling perfection with application-optimized contrasts using different flip-angle evolutions, and T2w two-dimensional (2D) turbo spin-echo sequences. Two observers independently performed two olfactory bulb volumetric studies per bulb and sequence. Intraobserver and interobserver reliability was assessed using intraclass correlation coefficients. For the evaluation of reproducibility, concordance correlation coefficients were determined, and for repeatability and systematic biases, Bland-Altman plots were analyzed. Results: Intraclass correlation coefficient analysis of the specialized observer yielded almost perfect results for intraobserver reliability (0.94, 0.85, and 0.93 for the CISS, T2w 3D, and T2w 2D sequences, respectively). For the less experienced observer, the results were 0.86 0.78, and 0.74 for the CISS, T2w 3D, and T2w 2D sequences, respectively. Interobserver reliability showed almost perfect agreement for all sequences (0.92, 0.86, and 0.86, respectively). The CISS sequence yielded the highest concordance correlation coefficient (0.84), precision (0.85), and accuracy (0.99). Bland-Altman plot analyses revealed the lowest repeatability coefficients for the T2w 2D sequence. Volumetric measurements of T2w 2D imaging showed systematically lower volumetric results compared to the CISS sequence (−22.7%) and the T2w 3D sequence (−8.3%). Conclusions: Comparison of three imaging sequences for olfactory bulb volumetry yielded the best values for the CISS sequence in terms of intraobserver and interobserver reliability, reproducibility, accuracy, and precision. Given that even less experienced observers achieve almost perfect results, the CISS sequence is recommended for olfactory bulb volumetry. [Copyright &y& Elsevier]

Rationale and Objectives: The aim of this study was to evaluate the suitability of 3-T magnetic resonance imaging (MRI) for olfactory bulb volumetry, comparing image quality obtained using different sequences on the basis of physical characteristics in combination with observer performance. Materials and Methods: Twenty-two healthy volunteers (11 men, 11 women; mean age, 25 years) underwent 3-T MRI of the frontal skull base in this prospective study. Imaging was performed using a constructive interference in steady state (CISS) three-dimensional Fourier transformation sequence, a three-dimensional T2-weighted (3D-T2w) sequence, and a two-dimensional T2-weighted (2D-T2w) sequence. The relative performance of sequences was assessed using visual grading characteristic analysis. Interobserver agreement was assessed using κ statistics. For evaluation of physical image quality characteristics, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated and compared using Wilcoxon’s test. SNR and CNR measurements were correlated with visual grading results. Results: Visual grading characteristic analysis showed significantly better image quality ratings for the CISS sequence compared to the 3D-T2w and 2D-T2w sequence, and the 2D-T2w sequence performed significantly better compared to the 3D-T2w sequence (P < .001). Interobserver agreement was substantial (κ = 0.66–0.73). Wilcoxon’s test revealed significantly higher SNR and CNR values for the 2D-T2w sequence compared to the 3D-T2w and CISS sequences, and SNR and CNR values for the 3D-T2w sequence were significantly higher compared to those for the CISS sequence (P < .001 for each). Significant correlation between SNR and CNR and visual grading was found for the 3D-T2w sequence (SNR: ρ = 0.510, P = .015; CNR: ρ = 0.546, P = .009). Conclusions: High-resolution 3-T MRI resulted in excellent values for SNR and CNR, suggesting the appropriateness of the sequences for olfactory bulb MRI volumetry. Visual grading characteristic analysis revealed the CISS sequence to be the most suitable for olfactory bulb volumetry. [Copyright &y& Elsevier]

Abstract: Objective: Performance-enhancing substances are illicitly used in elite or amateur sports and may be obtained from the black market due to a cheaper and easier availability. Although various studies have shown that black market products frequently do not contain the declared substances, enormous amounts of illegally produced and/or imported drugs are confiscated from athletes or at customs with alarming results concerning the outcome of the analyses of the ingredients. This case report describes the identification of His-tagged Long-R3-IGF-I, which is usually produced for biochemical studies, in an injection vial. Design: The ingredients were isolated by immunoaffinity purification and identified by nano-UPLC, high-resolution/high accuracy mass spectrometry of the intact and trypsinated substance and by an enzyme-linked immunosorbent assay. Results: (Tandem) mass spectra characterized the protein as Long-R3-IGF-I with a His6-tag attached to the C-terminus by the linker amino acids Leu–Glu. Conclusion: His-tags are commonly added to proteins during synthesis to allow a convenient and complete purification of the final product and His-tags are subsequently removed by specific enzymes when being attached to the N-terminus. The effects of His-tagged Long-R3-IGF-I in humans have not been elucidated or described and the product may rather be a by-product from biochemical studies than synthesized for injection purposes. [ABSTRACT FROM AUTHOR]

Rationale and Objectives: Enhancement characteristics after administration of a contrast agent are regarded as a major criterion for differential diagnosis in magnetic resonance mammography (MRM). However, no consensus exists about the best measurement method to assess contrast enhancement kinetics. This systematic investigation was performed to compare visual estimation with manual region of interest (ROI) and computer-aided diagnosis (CAD) analysis for time curve measurements in MRM. Materials and Methods: A total of 329 patients undergoing surgery after MRM (1.5 T) were analyzed prospectively. Dynamic data were measured using visual estimation, including ROI as well as CAD methods, and classified depending on initial signal increase and delayed enhancement. Results: Pathology revealed 469 lesions (279 malignant, 190 benign). Kappa agreement between the methods ranged from 0.78 to 0.81. Diagnostic accuracies of 74.4% (visual), 75.7% (ROI), and 76.6% (CAD) were found without statistical significant differences. Conclusions: According to our results, curve type measurements are useful as a diagnostic criterion in breast lesions irrespective of the method used. [Copyright &y& Elsevier]

Rationale and Objectives: The identification of the most suspect enhancing part of a lesion is regarded as a major diagnostic criterion in dynamic magnetic resonance mammography. Computer-aided diagnosis (CAD) software allows the semi-automatic analysis of the kinetic characteristics of complete enhancing lesions, providing additional information about lesion vasculature. The diagnostic value of this information has not yet been quantified. Materials and Methods: Consecutive patients from routine diagnostic studies (1.5 T, 0.1 mmol gadopentetate dimeglumine, dynamic gradient-echo sequences at 1-minute intervals) were analyzed prospectively using CAD. Dynamic sequences were processed and reduced to a parametric map. Curve types were classified by initial signal increase (not significant, intermediate, and strong) and the delayed time course of signal intensity (continuous, plateau, and washout). Lesion enhancement was measured using CAD. The most suspect curve, the curve-type distribution percentage, and combined dynamic data were compared. Statistical analysis included logistic regression analysis and receiver-operating characteristic analysis. Results: Fifty-one patients with 46 malignant and 44 benign lesions were enrolled. On receiver-operating characteristic analysis, the most suspect curve showed diagnostic accuracy of 76.7 ± 5%. In comparison, the curve-type distribution percentage demonstrated accuracy of 80.2 ± 4.9%. Combined dynamic data had the highest diagnostic accuracy (84.3 ± 4.2%). These differences did not achieve statistical significance. With appropriate cutoff values, sensitivity and specificity, respectively, were found to be 80.4% and 72.7% for the most suspect curve, 76.1% and 83.6% for the curve-type distribution percentage, and 78.3% and 84.5% for both parameters. Conclusions: The integration of whole-lesion dynamic data tends to improve specificity. However, no statistical significance backs up this finding. [Copyright &y& Elsevier]

Abstract: This study investigates the influence of pelvic tilt on conventional alignment of acetabular cups. Cementless cups were aligned into a synthetic replica of the pelvis 300 times at different pelvic tilts. At +10° pelvic tilt, average cup inclination was 46.2° (32° to 65°; ±7.0°), and average cup anteversion was 19.8° (4° to 37°; ±9.1°). At neutral pelvic tilt, inclination was 44.5° (28° to 59°; ±7.2°), and anteversion was 15.6° (−5° to 33°; ±8.1°). At −10° pelvic tilt, inclination was 42.6° (25° to 61°; ±7.2°), and anteversion was 10.5° (−10° to 37°; ±12.2°). Overall, 50% of the cups were positioned outside the safe zone: 46% in pelvic inclination, 42% in neutral position, and 63% in pelvic reclination (P = .007). This study shows the considerable inaccuracies of conventional cup implantation by experienced and trainee surgeons and shows the influence of pelvic tilt on acetabular cup alignment. [Copyright &y& Elsevier]

Polyunsaturated fatty acids (PUFAs), including essential fatty acids linoleic and α-linolenic acid and derived long chain and very long chain ω3-and ω6-polyunsaturated fatty acids, are vital structures in mammalian membrane systems and signaling molecules, pivotal in brain development, lipid, and energy metabolism and in female and male fertility during human evolution. Numerous nutritional studies suggest imbalance of PUFA metabolism as a critical factor in the pathogenesis of several human lifestyle diseases: dyslipoproteinemia, obesity, cardiovascular and neurodegenerative diseases, and infertility. The lack of unbiased animal models impedes molecular interpretation of the role of synthesized and dietary supplied PUFAs in these conditions. In this study, we used a Δ6 fatty acid desaturase (FADS2) deficient mouse mutant lacking key enzyme activity in the biosynthesis of ω3-and ω6-PUFAs from EFAs to address the molecular role of PUFAs in female and male fertility. Infertility is a hallmark of the pleiotropic but auxotrophic fads2 −/− phenotype and is therefore helpful for stringent dietary studies on the role of individual PUFAs. Feeding regimens: Age- and gender-matched infertile fads2−/− mice were maintained on defined diets, normal diet containing essential fatty acids, and supplemented with ω6-arachidonic acid, ω3-docosahexaenoic acid, and arachidonic/docosahexaenoic acid, starting (a) after weaning and (b) initiated in 4-month-old female and male fads2 −/− mice. Phospho- and sphingolipidomes of ovarian and testicular membrane lipid bilayers in each cohort were established and the impact on the expression and topology of membrane marker proteins, membrane morphology, germ cell development, and female and male fertility in the respective cohorts was elaborated. PUFA synthesis deficiency caused a halt to folliculogenesis, atresia of oocytes, and infertility of fads2 −/− female mice. A PUFA-deficient membrane lipid bilayer core structure led to the disassembly of the gap junction network of the follicular granulosa cells. In fads2 −/− testis, the blood-testis barrier was disrupted and spermatogenesis arrested, leading to infertility. Sustained supply of combined AA and DHA remodeled the PUFA-deficient ovarian and testicular membrane lipidomes, facilitating the reassembly of the functional gap junction network for regular ovarian cycles and the reconstitution of the blood-testis barrier in Sertoli cells, reconstituting fertility not only in developing newborns, but surprisingly also in adult infertile fads2 −/− mice. These findings demonstrate the previously unrecognized membrane structure-based molecular link between nutrient ω3-and ω6-PUFAs, gonadal membrane structures, and female and male fertility and might foster studies of the pivotal role of dietary PUFAs in human fertility. Image 1 • PUFA-depletion disrupts membrane lipid scaffolds of ovarian GJ- and TJ-complexes of the testicular BTB • Nutrient AA/DHA reconstitute the gonadal membrane bilayer architecture in auxotrophioc fads2-/- mice • AA/DHA replenished lipid-bilayers promote the assembly of follicular GJ- and BTB-protein complexes in fads2-/- mice • Nutrient AA/DHA release arrest of oo- and spermatogenesis, restoring fertility of newborn and adult fads2-/- mice [ABSTRACT FROM AUTHOR]

A 34-year old male was found breathless and panting at home by his girlfriend three hours after a gym workout. Minutes later, he collapsed and died. Autopsy, histological and chemical analyses were conducted. The examination of the heart showed left ventricular hypertrophy, while the right coronary artery showed only a small vascular lumen (3 mm in diameter), due to its anatomical structure. In femoral blood concentrations of approx. 1 μg/L clenbuterol, approx. 56 μg/L stanozolol and approx. 8 μg/L metandienone, with trenbolone (

Background: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and is increasing, primarily among women. Underdiagnosis is common, and because of the heterogeneous disease characteristics, molecular markers of specific disease phenotypes and more efficacious treatment regimens are urgently needed. Objective: In this study the soluble proteome of bronchoalveolar lavage cells, primarily consisting of macrophages, was investigated with the aim of identifying phenotypic differences in early disease development. Methods: Two-dimensional difference gel electrophoresis was used for relative quantification of protein levels, and multivariate modeling was applied to identify proteins of interest that were subsequently identified by means of liquid chromatography–mass spectrometry. Results: Significant gender differences were unveiled, with numerous alterations in the bronchoalveolar lavage cell proteome occurring in female but not male patients with COPD. Specifically, a subset of 19 proteins provided classification of female healthy smokers from female patients with COPD with 78% predictive power. Subsequent pathway analyses linked the observed alterations to downregulation of the lysosomal pathway and upregulation of the oxidative phosphorylation pathway, possibly linking dysregulation of macroautophagy to a female-dominated COPD disease phenotype. Conclusion: This investigation makes an important contribution to the elucidation of putative molecular mechanisms underlying gender-based differences in the pathophysiology of COPD, linking alterations of specific molecular pathways to previously observed gender differences in clinical COPD phenotypes. Furthermore, these results stress the importance of the gender-specific search for biomarkers, diagnosis, and treatment in COPD. [Copyright &y& Elsevier]

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation. In this study, a novel multi-target approach based on liquid chromatography and high resolution/high accuracy mass spectrometry is presented to screen for more than 200 analytes of various classes of doping agents far below the required detection limits in sports drug testing. Here, classic groups of drugs as diuretics, stimulants, β 2 -agonists, narcotics and anabolic androgenic steroids as well as various newer target compounds like hypoxia-inducible factor (HIF) stabilizers, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), plasma volume expanders and other doping related compounds, listed in the 2016 WADA prohibited list were implemented. As a main achievement, growth hormone releasing peptides could be implemented, which chemically belong to the group of small peptides (<2 kDa) and are commonly determined by laborious and time-consuming stand-alone assays. The assay was fully validated for qualitative purposes considering the parameters specificity, robustness (rRT: <2%), intra- (CV: 1.7–18.4 %) and inter-day precision (CV: 2.3–18.3%) at three concentration levels, linearity (R 2 > 0.99), limit of detection (0.1–25 ng/mL; 3′OH-stanozolol glucuronide: 50 pg/mL; dextran/HES: 10 μg/mL) and matrix effects. [ABSTRACT FROM AUTHOR]

Chlorazanil (Ordipan, N -(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N -(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N -(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance. [ABSTRACT FROM AUTHOR]

[Display omitted] • Probing for metabolites substantially improves the overall anti-doping work. • Long-term metabolites and long-term storage of doping control samples have considerably improved anti-doping retrospectivity. • Metabolic pathways of lower molecular mass doping agents are traceable by isotope-ratio mass spectrometry. • Metabolic pathways of peptidic doping agents are traceable by stable isotope labeling and HRMS. • Drug elimination profiles support result managing processes with regards to (presumed) time points of drug administration. Probing for evidence of the administration of prohibited therapeutics, drugs and/or drug candidates as well as the use of methods of doping in doping control samples is a central assignment of anti-doping laboratories. In order to accomplish the desired analytical sensitivity, retrospectivity, and comprehensiveness, a considerable portion of anti-doping research has been invested into studying metabolic biotransformation and elimination profiles of doping agents. As these doping agents include lower molecular mass drugs such as e.g. stimulants and anabolic androgenic steroids, some of which further necessitate the differentiation of their natural/endogenous or xenobiotic origin, but also higher molecular mass substances such as e.g. insulins, growth hormone, or siRNA/anti-sense oligonucleotides, a variety of different strategies towards the identification of employable and informative metabolites have been developed. In this review, approaches supporting the identification, characterization, and implementation of metabolites exemplified by means of selected doping agents into routine doping controls are presented, and challenges as well as solutions reported and published between 2010 and 2020 are discussed. [ABSTRACT FROM AUTHOR]

• SI changes are unreliable parameters to evaluate gadolinium deposition in the brain. • SI changes are also unreliable to identify factors that affect gadolinium deposition. • No factor had a consistent influence on SI changes for either reader. To evaluate the reliability of signal intensity (SI) changes in the basal ganglia as a supposed indicator of gadolinium deposition in the brain after repetitive application of gadolinium-based contrast agents (GBCAs) in a pediatric neuro-oncological collective. One hundred and eight neuropediatric patients (54 male, 54 female, 0–17 years old), with repetitive GBCA-enhanced cranial MRIs between 2003 and 2017, were retrospectively analyzed. Two radiologists measured SI in the nucleus dentatus (ND), globus pallidus (GP), thalamus (T), and the pons (P). The N D P and G P T ratio were calculated. An intraclass correlation coefficient, and multiple linear regressions with subsequent stepwise backward variable selection were performed to evaluate the influence of gender, patient's age at the first MRI, time interval between the first and last MRI, linear or macrocyclic GBCAs, residual pathology, treatments, and magnet field strengths. The inter-reader agreement was good for G P T and N D P in the whole collective (ICC = 0.837 and ICC = 0.793) and for children >2 years of age (ICC = 0.874 and ICC = 0.790), but poor to moderate for children ≤2 years of age (ICC = 0.397 and ICC = 0.748). The intra-reader agreement was good (ICC = 0.910 and ICC = 0.882). An SI increase was only observed for both readers in G P T (p = 0.003, or p < 0.001). None of the considered cofactors showed a consistent effect on SI changes for either readers or regions. Measurements of SI changes in the basal ganglia are not a reliable parameter with which to evaluate or estimate gadolinium deposition in the brain or to identify suspicious influential factors after repeated GBCA applications. [ABSTRACT FROM AUTHOR]

Aim Posttransplantation diabetes mellitus (PTDM) is a common complication after renal transplantation leading to increased cardiovascular morbidity and mortality. In subjects with type 2 diabetes (T2DM) increased glycemic variability and poor glycemic control have been associated with cardiovascular complications. We therefore aimed at determining glycemic variability and glycemic control in subjects with PTDM in comparison to T2DM subjects. Methods In this observational study we analyzed 10 transplanted subjects without diabetes (Control), 10 transplanted subjects with PTDM, and 8 non-transplanted T2DM subjects using Continuous Glucose Monitoring (CGM). Several indices of glycemic control quality and variability were computed. Results Many indices of both glycemic control quality and variability were different between control and PTDM subjects, with worse values in PTDM. The indices of glycemic control, such as glucose mean, GRADE and M-value, were similar in PTDM and T2DM, but some indices of glycemic variability, that is CONGA, lability index and shape index, showed a markedly higher (i.e., worse) value in T2DM than in PTDM ( P value range: 0.001–0.035). Conclusions Although PTDM and T2DM subjects showed similar glycemic control quality, glycemic variability was significantly higher in T2DM. These data underscore potential important pathophysiological differences between T2DM and PTDM indicating that increased glycemic variability may not be a key factor for the excess cardiovascular mortality in patients with PTDM. [ABSTRACT FROM AUTHOR]

Differentiation of insulin analogues is required in forensic and clinical toxicology as well as in sports doping control. Immunoassay results provide only weak evidence for exogenous administration of insulin, as concentrations cannot be reliably interpreted and specific information on the insulin species remains unknown. In post-mortem blood, insulin degrades rapidly. In this study, improved methodology consisting of precipitation of proteins, immunoaffinity purification and liquid chromatography coupled to high resolution/high accuracy mass spectrometry were applied to post-mortem vitreous humour. Ten successive cases with a post-mortem interval from four to ten days were investigated for insulin analogues. The cause of death in these cases was connected with diabetes and its complications, as well as with chronic cardiovascular disease, alcoholism and cancer. In all cases, the manner of death was natural (disease). Insulin was positively detected in post-mortem vitreous humour in three cases out of ten by mass spectrometry. In two cases, the method revealed the long-acting insulin glargine (Lantus) metabolite M2 (DesB31-32 Lantus), and human insulin was detected in one case. The findings were in agreement with the documented history of insulin medication. No other obvious reason could be found for the failure of detecting insulins in the other cases than insulin degradation during the lengthy postmortem interval. Vitreous humour is still a most prospective specimen for detection of insulin analogues post-mortem. [ABSTRACT FROM AUTHOR]