TREM2+ and interstitial-like macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The immunopathological mechanisms driving the development of severe COVID-19 remain poorly defined. Here, we utilize a rhesus macaque model of acute SARS-CoV-2 infection to delineate perturbations in the innate immune system. SARS-CoV-2 initiates a rapid infiltration of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generate a longitudinal scRNA-Seq dataset of airway cells, and map these subsets to corresponding populations in the human lung. SARS-CoV-2 infection elicits a rapid recruitment of two macrophage subsets: CD163+MRC1-, and TREM2+ populations that are the predominant source of inflammatory cytokines. Treatment with baricitinib (Olumiant®), a JAK1/2 inhibitor is effective in eliminating the influx of non-alveolar macrophages, with a reduction of inflammatory cytokines. This study delineates the major lung macrophage subsets driving airway inflammation during SARS-CoV-2 infection.

Ambient ionization mass spectrometry applied to new psychoactive substance analysis.

In the past decade a plethora of drugs with similar effects to controlled psychoactive drugs, like cannabis, amfetamine (amphetamine), or lysergic acid diethylamide, have been synthesized. These drugs can collectively be classified under the term new psychoactive substances (NPS) and are used for recreational purposes. The novelty of the substances, alongside the rapid rate of emergence and structural variability, makes their detection as well as their legal control highly challenging, increasing the demand for rapid and easy-to-use analytical techniques for their detection and identification. Therefore, interest in ambient ionization mass spectrometry applied to NPS has grown in recent years, which is largely because it is relatively fast and simple to use and has a low operating cost. This review aims to provide a critique of the suitability of current ambient ionization techniques for the analysis of NPS in the forensic and clinical toxicology fields. Consideration is given to analytical performance and ease of implementation, including ionization efficiency, selectivity, sensitivity, quantification, analyte chemistry, molecular coverage, validation, and practicality.

Quantification of insulin-like growth factor-I by liquid chromatography-mass spectrometry.

We describe three methods of sample preparation for the liquid chromatography coupled with mass spectrometric measurement of insulin-like growth factor-I concentration in blood. One method involves trypsin digestion, the second involves intact protein quantification and the third method is a combination of the first two. Step-by-step directions are provided for sample collection and handling including transport, storage conditions as well as detailed instructions for preparation for analysis, which can be modified for larger or smaller sample volumes as needed. A fully 15 N-labelled internal standard is used and the merits of a single-point calibrator are discussed. Example instrumental conditions are presented for both intact and digest methods.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.

A miniaturized passive sampling-based workflow for monitoring chemicals of emerging concern in water.

The miniaturization of a full workflow for identification and monitoring of contaminants of emerging concern (CECs) is presented. Firstly, successful development of a low-cost small 3D-printed passive sampler device (3D-PSD), based on a two-piece methacrylate housing that held up to five separate 9 mm disk sorbents, is discussed. Secondly, a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method reduced the need for large scale in-laboratory apparatus, solvent, reagents and reference material quantities for in-laboratory passive sampler device (PSD) calibration and extraction. Using hydrophilic-lipophilic balanced sorbents, sampling rates (Rs) were determined after a low 50 ng L-1 exposure over seven days for 39 pesticides, pharmaceuticals, drug metabolites and illicit drugs over the range 0.3 to 12.3 mL day-1. The high sensitivity LC-MS/MS method enabled rapid analysis of river water using only 10 µL of directly injected sample filtrate to measure occurrence of 164 CECs and sources along 19 sites on the River Wandle, (London, UK). The new 3D-PSD was then field-tested over seven days at the site with the highest number and concentration of CECs, which was down-river from a wastewater treatment plant. Almost double the number of CECs were identified in 3D-PSD extracts across sites in comparison to water samples (80 versus 42 CECs, respectively). Time-weighted average CEC concentrations ranged from 8.2 to 845 ng L-1, which were generally comparable to measured concentrations in grab samples. Lastly, high resolution mass spectrometry-based suspect screening of 3D-PSD extracts enabled 113 additional compounds to be tentatively identified via library matching, many of which are currently or are under consideration for the EU Watch List. This miniaturized workflow represents a new, cost-effective, and more practically efficient means to perform passive sampling chemical monitoring at a large scale. SYNOPSIS: Miniaturized, low cost, multi-disk passive samplers enabled more efficient multi-residue chemical contaminant characterization, potentially for large-scale monitoring programs.

Combined statistical decision limits based on two GH-2000 scores for the detection of growth hormone misuse.

The growth hormone-2000 biomarker method, based on the measurements of insulin-like growth factor-I and the amino-terminal pro-peptide of type III collagen, has been developed as a powerful technique for the detection of growth hormone misuse by athletes. Insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen are combined in gender-specific formulas to create the growth hormone-2000 score, which is used to determine whether growth hormone has been administered. To comply with World Anti-Doping Agency regulations, each analyte must be measured by two methods. Insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen can be measured by a number of approved methods, each leading to its own growth hormone-2000 score. Single decision limits for each growth hormone-2000 score have been introduced and developed by Bassett, Erotokritou-Mulligan, Holt, Böhning and their co-authors in a series of papers. These have been incorporated into the guidelines of the World Anti-Doping Agency. A joint decision limit was constructed based on the sample correlation between the two growth hormone-2000 scores generated from an available sample to increase the sensitivity of the biomarker method. This paper takes this idea further into a fully developed statistical approach. It constructs combined decision limits when two growth hormone-2000 scores from different assay combinations are used to decide whether an athlete has been misusing growth hormone. The combined decision limits are directly related to tolerance regions and constructed using a Bayesian approach. It is also shown to have highly satisfactory frequentist properties. The new approach meets the required false-positive rate with a pre-specified level of certainty.

Procollagen type III amino-terminal propeptide and insulin-like growth factor I as biomarkers of growth hormone administration.

The acceptance in 2012 by the World Anti-Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on procollagen type III amino-terminal propeptide (P-III-NP) and insulin-like growth factor I (IGF-I) was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti-doping standards, where the strict liability principle applies, is discussed. An alternative WADA-accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half-life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When a liquid chromatography coupled mass spectrometry (LC-MS) method became a reality for the measurement of IGF-I, more consistency of results was assured. Measurement of P-III-NP is still dependent on immunoassays although work is underway to develop an LC-MS method. The promised long-term detection time for the biomarker assay does not appear to have been realised in practice, and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P-III-NP and IGF-I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the growth hormone (GH) score would mandate an investigation and possible action for hGH administration.

CCR2 Signaling Restricts SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant ® ), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. ONE SENTENCE SUMMARY: Multi-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib.

CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection.

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

Rapid direct analysis of river water and machine learning assisted suspect screening of emerging contaminants in passive sampler extracts.

A novel and rapid approach to characterise the occurrence of contaminants of emerging concern (CECs) in river water is presented using multi-residue targeted analysis and machine learning-assisted in silico suspect screening of passive sampler extracts. Passive samplers (Chemcatcher®) configured with hydrophilic-lipophilic balanced (HLB) sorbents were deployed in the Central London region of the tidal River Thames (UK) catchment in winter and summer campaigns in 2018 and 2019. Extracts were analysed by; (a) a rapid 5.5 min direct injection targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for 164 CECs and (b) a full-scan LC coupled to quadrupole time of flight mass spectrometry (QTOF-MS) method using data-independent acquisition over 15 min. From targeted analysis of grab water samples, a total of 33 pharmaceuticals, illicit drugs, drug metabolites, personal care products and pesticides (including several EU Watch-List chemicals) were identified, and mean concentrations determined at 40 ± 37 ng L-1. For targeted analysis of passive sampler extracts, 65 unique compounds were detected with differences observed between summer and winter campaigns. For suspect screening, 59 additional compounds were shortlisted based on mass spectral database matching, followed by machine learning-assisted retention time prediction. Many of these included additional pharmaceuticals and pesticides, but also new metabolites and industrial chemicals. The novelty in this approach lies in the convenience of using passive samplers together with machine learning-assisted chemical analysis methods for rapid, time-integrated catchment monitoring of CECs.

Determination of anabolic steroids in dried blood using microsampling and gas chromatography-tandem mass spectrometry: Application to a testosterone gel administration study.

Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL-1 in 20 µL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.

In Vitro Phase I Metabolic Profiling of the Synthetic Cannabinoids AM-694, 5F-NNEI, FUB-APINACA, MFUBINAC, and AMB-FUBINACA.

Synthetic cannabinoids (SCs) constitute one of the most rapidly expanding class of new psychoactive substances. SCs pose a health threat to the individual and to the public due to their central (psychoactive) and peripheral effects. Their pharmacology and toxicology are poorly understood, and the substances can be unexpectedly toxic and harmful. The metabolism of SCs is also relevant in clinical and forensic toxicology as SCs are excreted in urine mostly as their metabolites. Thus, SC metabolites are widely used as markers for identifying SC intake. Herein, we used human liver microsome systems to study the in vitro phase I metabolic profiling of five SCs, namely AM-694, 5F-NNEI, FUB-APINACA, MFUBINAC, and AMB-FUBINACA. The metabolites were detected and structurally elucidated by liquid chromatography-high resolution mass spectrometry. The main metabolic pathway of AM-694 (benzoyl-indole SC) is oxidative defluorination; 5F-NNEI (naphthyl-indole carboxamide SC) follows amide hydrolysis and monohydroxylation at the naphthyl moiety. However, indazole carboxamide substituted with an adamantyl group, such as FUB-APINACA, is likely to produce (isomeric) hydroxylation of the adamantyl group as the main metabolite species. For the substrates that contain ester bonds in their structure, like MFUBINAC and AMB-FUBINACA, the ester hydrolysis metabolite is predominant.

Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry.

BACKGROUND: Insulin-like growth factor-I (IGF-1) is measured mainly by immunoassay for the diagnosis and treatment of growth hormone (GH) disorders, and to detect misuse of GH in sport. Immunoassays often have insufficient inter-laboratory agreement, especially between commercial kits. Over the expected range of IGF-1 in blood (∼50-500 ng/mL), in an inter-laboratory study we previously established a measurement imprecision of 11% (%CV) for the digested protein analyzed by LC-MS. Measuring intact IGF-1 by LC-MS should be simpler. However, no inter-laboratory agreement has been published. METHODS: Intact and trypsin-digested IGF-1 in 32 serum samples from healthy volunteers and human growth hormone administration studies were analyzed by LC-MS using different instruments in five laboratories, as well as by immunoassay in a single laboratory. Another 100 samples were analyzed for IGF-1, both intact and after trypsin-digestion, in each laboratory by LC-MS. The statistical relationship between measurements and the imprecision of each assay group was assessed. RESULTS: An intra-laboratory variability of 2-4% CV was obtained. Inter-laboratory variability was greater at 14.5% CV. Orthogonal regression of intact versus trypsin-digestion methods (n = 646) gave a slope of 1.01 and intercept of 2.05 ng/mL. CONCLUSIONS: LC-MS measurements of IGF-1 by intact and trypsin-digestion methods are not statistically different and each is similar to immunoassay. The two LC-MS approaches may be used interchangeably or together to eliminate concerns regarding an immunoassay IGF-1 measurement. Because intact and digested IGF-1 measurements generally agreed within 20% of each other, we propose this as a criterion of assay acceptability.

Evaluation of combined sewer overflow impacts on short-term pharmaceutical and illicit drug occurrence in a heavily urbanised tidal river catchment (London, UK).

The occurrence of pharmaceutical and illicit drug residues potentially arising from combined sewer overflows (CSOs) in the Central London portion of the Thames Estuary is presented. Approximately 39 million tonnes of untreated sewage enter the River Thames at 57 CSO points annually. Differential analysis of influents and effluents in a major wastewater treatment plant identified seven potential drug-related CSO markers based on removal rates. Three were present in influent at concentrations >1 µg L-1 (caffeine, cocaine and benzoylecgonine). During dry weather, analysis of hourly samples of river water revealed relatively consistent concentrations for most drugs, including CSO markers, over a tidal cycle. River water was monitored over a week in January and July and then daily across six consecutive weeks in November/December 2014. Out of 31 compounds monitored, 27 drug residues were determined in the River Thames and, combined, ranged between ~1000-3500 ng L-1. Total drug concentration generally declined during extended periods of drier weather. For CSO markers, short-term increases in caffeine, cocaine and benzoylecgonine concentration were observed ~24 h after CSO events (especially those occurring at low tide) and generally within one order of magnitude. Timings of elevated occurrence also correlated well with ammonium ion and dissolved oxygen data following CSOs. This work also represents an important study of pharmaceutical occurrence before a major 'Super Sewer' infrastructure upgrade in London aiming to reduce CSOs by 95%.

IRMS delta values (13 C) of nandrolone and testosterone products available in the UK: Implications for anti-doping.

Anabolic androgenic steroids (AAS) are the most widely abused class of drugs by athletes and thus represent a significant problem to the anti-doping community. Confirmation of a doping violation for AAS cannot always be based on their presence alone due to the endogenous production of some steroids. Both testosterone (and its metabolites) and the major diagnostic metabolite of nandrolone (19-norandrosterone) are produced endogenously. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is used in such cases to differentiate between the administration of a synthetic preparation and endogenous steroid production by measurement of their differing carbon isotope (13 C/12 C) ratio. The purpose of this study was to investigate the availability of steroid preparations in the UK with a 13 C content analytically indistinguishable from that of endogenous steroids. Fourteen preparations containing nandrolone (n = 9) and testosterone (n = 5) were analyzed. The δ13 C values were determined using GC-C-IRMS and the identity of the steroid preparations was confirmed using gas chromatography-mass spectrometry (GC-MS). Ten steroid preparations displayed δ13 C values within the range expected for synthetic steroids (less than -27‰). However, four nandrolone preparations displayed δ13 C values that overlap with the values considered to be endogenous in origin (range: -26 to -16‰). Misuse of these preparations could prevent the confirmation of nandrolone administration using GC-C-IRMS in anti-doping cases.

A correction to the age-adjustment of the GH-2000 score used in the detection of growth hormone misuse.

OBJECTIVE: The GH-2000 biomarker test has been introduced by the World Anti-Doping Agency as a method of detecting growth hormone misuse in professional sport. The test involves the measurement insulin-like growth factor-I and the amino-terminal pro-peptide of type III collagen (P-III-NP) which increase in a dose-dependent manner in response to GH. These measurements are combined in sex specific formulae that include an age adjustment. The original age adjustment overcorrects the effect of age in male athletes and could potentially place older men at a disadvantage. The purpose of this note is to investigate the performance of a previously suggested correction term in two new and larger data sets. RESULTS: The GH-2000 score was calculated for 7307 samples obtained from 15 accredited WADA laboratories in 2017 and 3916 samples measured at Drug Control Centre, King's College London, UK between 2013 and 2017. The GH-2000 scores were investigated for positive age effects using standard regression modelling. As previously, all analyses confirmed a positive age effect. Applying the earlier suggested correction term of 0.032 × age showed a significant over-correction leading to a negative association of the GH-2000 score with age. We now suggest a smaller age correction of 0.020 × age, which corresponds to the smallest effect found in the earlier studies.

Why do endocrine profiles in elite athletes differ between sports?

BACKGROUND: Endocrine profiles have been measured on blood samples obtained immediately post-competition from 693 elite athletes from 15 Olympic Sports competing at National or International level; four were subsequently excluded leaving 689 for the current analysis. METHODS: Body composition was measured by bioimpedance in a sub-set of 234 (146 men and 88 women) and from these data a regression model was constructed that enabled 'estimated' lean body mass and fat mass to be calculated on all athletes. One way ANOVA was used to assess the differences in body composition and endocrine profiles between the sports and binary logistical regression to ascertain the characteristic of a given sport compared to the others. RESULTS: The results confirmed many suppositions such as basketball players being tall, weightlifters short and cross-country skiers light. The hormone profiles were more surprising with remarkably low testosterone and free T3 (tri-iodothyronine) in male powerlifters and high oestradiol, SHBG (sex hormone binding globulin) and prolactin in male track and field athletes. Low testosterone concentrations were seen 25.4% of male elite competitors in 12 of the 15 sports and high testosterone concentrations in 4.8% of female elite athletes in 3 of the 8 sports tested. Interpretation of the results is more difficult; some of the differences between sports are at least partially due to differences in age of the athletes but the apparent differences between sports remain significant after adjusting for age. The prevalence of 'hyperandrogenism' (as defined by the IAAF (International Association of Athletics Federations) and IOC (International Olympic Committee)) amongst this cohort of 231 elite female athletes was the highest so far recorded and the very high prevalence of 'hypoandrogenism' in elite male athletes a new finding. CONCLUSIONS: It is unclear whether the differences in hormone profiles between sports is a reason why they become elite athletes in that sport or is a consequence of the arduous processes involved. For components of body composition we know that most have a major genetic component and this may well be true for endocrine profiles.