Quantitation of Formoterol, Salbutamol, and Salbutamol-4′-O-Sulfate in Human Urine and Serum via UHPLC-MS/MS

The adrenergic beta-2 agonists formoterol and salbutamol are used for the treatment of asthma and COPD but are also misused in sports competitions. Therefore, they are included in WADA regulations. Both drugs are mainly excreted in urine after administration via inhalation. A four-armed, double-blind cross-over clinical trial was conducted involving endurance-trained participants (12 females and 12 males). Inhalation dosages of 36 μg formoterol, 1200 μg salbutamol, a combination of both, or a placebo were administered before exercise. Serum and urine were collected after exercise and 3 and 24 h after administration. Here, we show the successful quantitation of formoterol, salbutamol, and its phase II metabolite salbutamol-4′-O-sulfate in all urine and serum samples using ultra-high performance liquid chromatography–tandem mass spectrometry. In the serum analysis, results of up to 14.2 pg/mL formoterol, 10.0 ng/mL salbutamol, and 21.4 ng/mL salbutamol-4′-O-sulfate (calculated as salbutamol equivalent) were found. In urine, maximum concentrations (after deglucuronidation) were 17.2 ng/mL formoterol, 948.5 ng/mL salbutamol, and 2738.5 ng/mL salbutamol-4′-O-sulfate. Sex-specific differences in serum concentrations as well as in urinary excretion were observed. The results pronounce the importance of the implementation and elucidation of phase II metabolites to quantitation methods in antidoping

Influence of Whole-Body Electrostimulation on the Deformability of Density-Separated Red Blood Cells in Soccer Players

Red blood cell nitric oxide synthase (RBC-NOS) dependent NO production positively affects RBC deformability which is known to improve oxygen supply to the working tissue. Whole-body electrostimulation (WB-EMS) has been shown to improve maximum strength, sprinting and jumping performance, and to increase deformability in elite soccer players during the season. The aim of the present study was to investigate whether WB-EMS affects RBC turnover which might affect overall deformability of circulating RBC by rejuvenation of the RBC population and if this might be related to improved endurance capacity. Thirty male field soccer players were assigned in either a WB-EMS group (EG, n = 10), a training group (TG, n = 10), or a control group (CG, n = 10). EG performed 3 × 10 squat jumps superimposed with WB-EMS twice per week in concurrent to 2–4 soccer training sessions and one match per week. TG only performed 3 × 10 squat jumps without EMS in addition to their soccer routine and the CG only performed the usual soccer training and match per week. Subjects were tested before (Baseline) and in week 7 (wk-7), with blood sampling before (Pre), 15–30 min after (Post), and 24 h after (24 h post) the training. Endurance capacity was determined before and directly after the training period. The key findings of the investigation indicate an increase in young RBC in the EG group along with improved overall RBC deformability, represented by decreased SS1/2:EImax Ratio. Analysis of the different RBC subfractions revealed improved RBC deformability of old RBC during study period. This improvement was not only observed in the EG but also in TG and CG. Changes in RBC deformability were not associated to altered RBC-NOS/NO signaling pathway. Endurance capacity remained unchanged during study period. In summary, the effect of WB-EMS on RBC physiology seems to be rather low and results are only in part comparable to previous findings. According to the lower training volume of the present study it can be speculated that the soccer specific training load in addition to the WB-EMS was too low to induce changes in RBC physiology

Lactate distribution in red blood cells and plasma after a high intensity running exercise in aerobically trained and untrained subjects

Introduction: To determine endurance capacity and to give specific training recommendations, blood lactate (LA) concentrations are frequently used in performance diagnostics. In blood, LA is stored in red blood cells (RBC) and in plasma. Higher LA uptake by RBC might lead to delayed muscle fatigue since RBC serve as a dilution space for LA and more LA can be taken up by plasma which is released from the working muscle. Therefore, the aim of this study was to investigate the distribution of lactate in plasma and RBC in aerobically well-trained athletes (AA) in comparison to an untrained control group (CG). Materials and Methods: 13 AA and 13 CG participated in this study and conducted a high intensive treadmill test consisting of 2x4 minutes of running at 95% of the maximal running velocity with an active break of 4 minutes. Venous blood was drawn before and after the test. LA was measured in whole blood, plasma and RBC. Further, the ratio (LARatio) was calculated using the following formula: LA of RBC / LA of plasma. Results: AA exhibit significantly higher values in VO2peak and maximum running velocity. After the running test, LA in whole blood, RBC and plasma is increased significantly in both groups. No interaction effect (group X time point) was observed in any parameter. Values of LARatio did not show any significant differences. Conclusion: This study showed that the LA distribution in RBC and plasma after a high intensity running test is very similar in well trained endurance athletes and in untrained control subjects. Hence, LA uptake by RBC cannot or only in part be seen as a contributor to aerobic athletic performance

The ELSA trial: single versus combinatory effects of non-prohibited beta-2 agonists on skeletal muscle metabolism, cardio-pulmonary function and endurance performance—study protocol for a randomized 4-way balanced cross-over trial

Background Asthma and/or airway hyper-responsiveness (AHR) are common in elite endurance athletes with a high prevalence rate of beta-2 adrenoreceptor (beta-2) agonists use. Nevertheless, there are data on dose-dependent ergogenic effects of beta-2 agonists suggesting increased muscle strength, endurance and neuromuscular performance. Therefore, most beta-2 agonists belong to the World Anti Doping Agency (WADA) list of prohibited substances and it is tempting to speculate that illegitimate use of beta-2 agonists might be a common practice to boost performance in competitive sports. It is currently unknown whether or not inhaled beta-2 agonists enhance performance by stimulatory effects in skeletal and cardiac muscle. Methods The ELSA trial is a double-blinded, placebo-controlled, randomized, balanced, four-way cross-over study. Study participants (n=24, 12 ♀, 12 ♂) complete four study arms (i.e. periods with treatment A, placebo; B, salbutamol; C, formoterol; D, formoterol + salbutamol) in random order after an initial preliminary testing session. Participants inhale the study medication 20 min before the 10-min time trial (TT; exercise performance test), where participants cycle 10 min at the highest possible workload. Cardiac output is measured continuously. A skeletal muscle biopsy is collected 3 h after the TT. Study endpoints include measures of skeletal muscle expression of nuclear receptors, hormones and cytokine levels, urinary and plasma concentrations of salbutamol and formoterol, circulating cardiac markers, cardiopulmonary function and exercise performance (average power and peak power during the TT). Blood and urine are collected and respiratory testing is performed 24 h post TT. Summary/conclusions This clinical trial evaluates the potential performance-enhancing effects of non-prohibited, not medically indicated inhaled short- and long-acting beta-2 agonists on skeletal muscle gene expression, endocrine regulation, cardiac biomarkers, cardiopulmonary function and acute endurance exercise performance. These data will be used by WADA to adapt the annually published list of prohibited substances (WADA 2021) and will be published in scientific journals

Acute Effects of Single Versus Combined Inhaled β2-Agonists Salbutamol and Formoterol on Time Trial Performance, Lung Function, Metabolic and Endocrine Variables

Background High prevalence rates of β2-agonist use among athletes in competitive sports makes it tempting to speculate that illegitimate use of β2-agonists boosts performance. However, data regarding the potential performance-enhancing effects of inhaled β2-agonists and its underlying molecular basis are scarce. Methods In total, 24 competitive endurance athletes (12f/12m) participated in a clinical double-blinded balanced four-way block cross-over trial to investigate single versus combined effects of β2-agonists salbutamol (SAL) and formoterol (FOR), to evaluate the potential performance enhancement of SAL (1200 µg, Cyclocaps, Pb Pharma GmbH), FOR (36 µg, Sandoz, HEXAL AG) and SAL + FOR (1200 µg + 36 µg) compared to placebo (PLA, Gelatine capsules containing lactose monohydrate, Pharmacy of the University Hospital Ulm). Measurements included skeletal muscle gene and protein expression, endocrine regulation, urinary/serum β2-agonist concentrations, cardiac markers, cardiopulmonary and lung function testing and the 10-min time trial (TT) performance on a bicycle ergometer as outcome variables. Blood and urine samples were collected pre-, post-, 3 h post- and 24 h post-TT. Results Mean power output during TT was not different between study arms. Treatment effects regarding lung function (p < 0.001), echocardiographic (left ventricular end-systolic volume p = 0.037; endocardial global longitudinal strain p < 0.001) and metabolic variables (e.g. NR4A2 and ATF3 pathway) were observed without any influence on performance. In female athletes, total serum β2-agonist concentrations for SAL and FOR were higher. Microarray muscle gene analysis showed a treatment effect for target genes in energy metabolism with strongest effect by SAL + FOR (NR4A2; p = 0.001). Of endocrine variables, follicle-stimulating hormone (3 h Post–Post-TT), luteinizing hormone (3 h Post–Pre-TT) and insulin (Post–Pre-TT) concentrations showed a treatment effect (all p < 0.05). Conclusions No endurance performance-enhancing effect for SAL, FOR or SAL + FOR within the permitted dosages compared to PLA was found despite an acute effect on lung and cardiac function as well as endocrine and metabolic variables in healthy participants. The impact of combined β2-agonists on performance and sex-specific thresholds on the molecular and cardiac level and their potential long-term performance enhancing or health effects have still to be determined. Trial registration: Registered at Eudra CT with the number: 2015-005598-19 (09.12.2015) and DRKS with number DRKS00010574 (16.11.2021, retrospectively registered)

Are Consumers Aware of Sustainability Aspects Related to Edible Insects? Results from a Study Involving 14 Countries

In recent years, edible insects have been suggested as an alternative food that is more sustainable compared with other sources of animal protein. However, knowledge about the sustainability aspects associated with this source of food may play a role in convincing consumers to adopt insects as part of their diet. In this context, the present study investigated the level of knowledge about the sustainability of edible insects in a group of people originating from 14 countries, with some naturally entomophagous and others not. To measure the knowledge, 11 items were selected and the scores obtained were tested with statistical tools (t-test for independent samples, analysis of variance—ANOVA) to search for differences according to sociodemographic and socioeconomic characteristics, geographical origin, and consumption habits of edible insects. The obtained results showed that, in general, knowledge is moderate, with the values of the average scores for the 11 items investigated ranging from 0.23 ± 0.99 to 0.66 ± 1.02, on a scale ranging from –2 (=very low knowledge) to 2 (=very high knowledge). The highest scores were found for items relating to the lower use of animal feed and lower emission of greenhouse gases required for the production of insects compared with beef. When investigating the differences between groups of participants, significant differences were generally found, revealing a trend for higher knowledge among males and young adults, for participants residing in urban areas and in countries such as Spain, Mexico, and Poland, and for participants with higher education levels and higher incomes. When testing the influence of consumption variables on the level of knowledge, the results showed a higher knowledge for participants who had already consumed insects or are willing to consume them. Finally, it was observed that higher knowledge was found for participants whose motivation to consume insects related to curiosity, a wish to preserve the planet, the gastronomic characteristics of insects, and their nutritional value. In conclusion, these results clearly indicate a very marked influence of a number of variables on the knowledge about the sustainability of edible insects, and this may be helpful to delineate strategies to effectively raise knowledge and eventually increase the willingness to consider insects as a more sustainable alternative to partially replace other protein foods, even in countries where this is a not a traditional practice.info:eu-repo/semantics/publishedVersio

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine1177 phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N5-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC

Iron: Managing obligations in higher-order concurrent separation logic

Precise management of resources and the obligations they impose, such as the need to dispose of memory, close locks, and release file handles, is hardÐespecially in the presence of concurrency, when some resources are shared, and different threads operate on them concurrently. We present Iron, a novel higher-order concurrentseparation logic that allows for precise reasoning about resources that are transferable among dynamically allocated threads. In particular, Iron can be used to show the correctness of challenging examples, where the reclamation of memory is delegated to a forked-off thread. We show soundness of Iron by means of a model ofIron, defined on top of the Iris base logic, and we use this model to prove that memory resources are accounted for precisely and not leaked. We have formalized all of the developments in the Coq proof assistant.Programming Language

Nitrite concentration and maximum RBC deformability (EI_max) in RBC after L-NIO incubation in comparison to untreated RBC.

<p>(A) RBC nitrite concentration was decreased in all subpopulations by L-NIO. Data are presented as Mean ± Standard Error of MEAN (n = 10). (B) EI_<b>max</b> values significantly decreased in subpopulations of 1.064 g/ml to 1.068 g/ml after L-NIO incubation. No decrease was observed in old subpopulations (1.070 g/ml and 1.076+ g/ml). Data are presented as Mean ± Standard Deviation (n = 10).</p