"Three-in-One": Ultrasensitive Lateral Flow Immunoassay Driven by Magnetic Enrichment and Photothermal Signal Amplification.

Conventional lateral flow immunoassay (LFIA) usually suffers from poor antimatrix interference, unsatisfactory sensitivity, and lack of quantitative ability for target analyte detection in food matrices. In response to these limits, here, multifunctional nanomaterial ZnFe2O4 nanoparticles (ZFOs) were developed and integrated into LFIA for powerful magnetic separation/enrichment and colorimetric/photothermal target sensing. Under optimum conditions, the detection for clenbuterol (CL) with magnetic enrichment achieves 9-fold higher sensitivity compared to that without enrichment and 162-fold higher sensitivity compared to that based on traditional colloidal golds. Attributing the improved performances of ZFOs, CL can be detected at ultralow levels in pork and milk with 10 min of immunoreaction time. The vLODs were 0.01 µg kg-1 for two modes, and the cutoff values of CL were about 5 and 3 µg kg-1, respectively. More importantly, the enrichment ZFO-mediated LFIA (ZE-LFIA) exhibits a similar limit of detection (LOD) in both buffer solution and food matrix, demonstrating a universal resistance to the food matrix. The multitudinous performance merits of this ZE-LFIA with high sensitivity, matrix tolerance, accuracy, and specificity have ensured a broad application potential for target detection of clenbuterol and can serve as an experience for other veterinary drug residues' detection.

Effects on cerebral blood flow after single doses of the ß2 agonist, clenbuterol, in healthy volunteers and patients with mild cognitive impairment or Parkinson's disease.

AIMS: Cerebral hypometabolism occurs years prior to a diagnosis of neurodegenerative diseases and coincides with reduced cerebral perfusion and declining noradrenergic transmission from the locus coeruleus. In pre-clinical models, ß-adrenoceptor (ß-AR) agonists increase cerebrocortical glucose metabolism, and may have therapeutic potential for neurodegenerative diseases. This study investigated the safety and effects on regional cerebral blood flow (rCBF) of the oral, brain-penetrant ß2-AR agonist, clenbuterol, in healthy volunteers (HV) and patients with mild cognitive impairment (MCI) or Parkinson's disease (PD). METHODS: This study evaluated the safety and effects on cerebral activity of the oral, brain-penetrant, ß2-AR agonist clenbuterol (20-160 µg) in healthy volunteers and patients with MCI or PD. Regional CBF, which is tightly coupled to glucose metabolism, was measured by arterial spin labelling MRI in 32 subjects (25 HV and 8 MCI or PD) across five cohorts. In some cohorts, low doses of nadolol (1-5 mg), a ß-AR antagonist with minimal brain penetration, were administered with clenbuterol to control peripheral ß2-AR responses. RESULTS: Significant, dose-dependent increases in rCBF were seen in multiple brain regions, including hippocampus, amygdala and thalamus, following the administration of clenbuterol to HVs (mean changes from baseline in hippocampal rCBF of -1.7%, 7.3%, 22.9%, 28.4% 3 h after 20, 40, 80 and 160 µg clenbuterol, respectively). In patients with MCI or PD, increases in rCBF following 80 µg clenbuterol were observed both without and with 5 mg nadolol (in hippocampus, 18.6%/13.7% without/with nadolol). Clenbuterol was safe and well-tolerated in all subjects; known side effects of ß2-agonists, including increased heart rate and tremor, were mild in intensity and were blocked by low-dose nadolol. CONCLUSIONS: The effects of clenbuterol on rCBF were evident both in the absence and presence of low-dose nadolol, suggesting central nervous system (CNS) involvement. Concomitant inhibition of the peripheral effects of clenbuterol by nadolol confirms that meaningful ß2-AR antagonism in the periphery was achieved without interrupting the central effects of clenbuterol on rCBF.

Machine learning powered CN-coordinated cobalt nanoparticles embedded cellulosic nanofibers to assess meat quality via clenbuterol monitoring.

The World Anti-Doping Agency (WADA) has prohibited the use of clenbuterol (CLN) because it induces anabolic muscle growth while potentially causing adverse effects such as palpitations, anxiety, and muscle tremors. Thus, it is vital to assess meat quality because, athletes might have positive test for CLN even after consuming very low quantity of CLN contaminated meat. Numerous materials applied for CLN monitoring faced potential challenges like sluggish ion transport, non-uniform ion/molecule movement, and inadequate electrode surface binding. To overcome these shortcomings, herein we engineered bimetallic zeolitic imidazole framework (BM-ZIF) derived N-doped porous carbon embedded Co nanoparticles (CN-CoNPs), dispersed on conductive cellulose acetate-polyaniline (CP) electrospun nanofibers for sensitive electrochemical monitoring of CLN. Interestingly, the smartly designed CN-CoNPs wrapped CP (CN-CoNPs-CP) electrospun nanofibers offers rapid diffusion of CLN molecules to the sensing interface through amine and imine groups of CP, thus minimizing the inhomogeneous ion transportation and inadequate electrode surface binding. Additionally, to synchronize experiments, machine learning (ML) algorithms were applied to optimize, predict, and validate voltametric current responses. The ML-trained sensor demonstrated high selectivity, even amidst interfering substances, with notable sensitivity (4.7527 µA/µM/cm2), a broad linear range (0.002-8 µM), and a low limit of detection (1.14 nM). Furthermore, the electrode exhibited robust stability, retaining 98.07% of its initial current over a 12-h period. This ML-powered sensing approach was successfully employed to evaluate meat quality in terms of CLN level. To the best of our knowledge, this is the first study of using ML powered system for electrochemical sensing of CLN.

Controlled release of Clenbuterol from a hydroxyapatite carrier for the treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder, and Aß aggregation is considered to be the central process implicated in its pathogenesis. Current treatments are faced by challenges such as serious side effects and reduced drug bioavailability. In this study, we developed a drug delivery system for intramuscular injection that uses cellular activity to achieve constant and long-term drug release. METHODS: Synthesized mesoporous hydroxyapatite (SHAP) was prepared via co-precipitation, and hydrophobic surface modification using stearic acid was then used to load clenbuterol by physical absorption, thus creating the drug delivery system. Clenbuterol release was achieved through cellular activity, with macrophage uptake triggering lysosome/endosome disruption, cytoplasmic release, extracellular exocytosis, and subsequent systemic circulation. RESULTS: We found that clenbuterol-loaded SHAP enabled sustained release for more than 2 weeks and effectively modulated inflammation, reduced Aß oligomer-induced toxicity, and prevented Aß aggregation. CONCLUSIONS: Our findings suggest that treatment with clenbuterol loaded in this SHAP delivery system could be a promising strategy for treating Alzheimer's disease.

Corticosterone injection into the basolateral amygdala before and after memory reactivation impairs the subsequent expression of fear memory in rats: An interaction of glucocorticoids and ß-adrenoceptors.

Glucocorticoid administration, before or after fear memory reactivation, impairs subsequent fear memory expression, but the underlying mechanisms are not well understood. The present study examined the role of basolateral amygdala (BLA) ß-adrenoceptors in the effects of intra-BLA corticosterone injection on fear memory in rats. Bilateral cannulae were implanted in the BLA of Wistar male rats. The rats were trained and tested using an inhibitory avoidance task (1 mA footshock for 3 s). Forty-eight hours after training, corticosterone (CORT, 5, 10, or 20 ng/0.5 µl/side) and the ß2-adrenoceptor agonist clenbuterol (CLEN, 10 or 20 ng/0.5 µl/side) or the ß-adrenoceptor antagonist propranolol (PROP, 250 or 500 ng/0.5 µl/side) were injected into the BLA before or right after memory reactivation (retrieval, Test 1). We performed subsequent tests 2 (Test 2), 5 (Test 3), 7 (Test 4), and 9 (Test 5) days after Test 1. The results demonstrated that CORT injection before Test 1 disrupted memory retrieval and reduced fear expression in Tests 2-5, possibly due to enhanced extinction or impaired reconsolidation. CORT injection after Test 1 also impaired reconsolidation and reduced fear expression in Tests 2-5. CLEN prevented, but PROP exacerbated, the effects of CORT on fear expression. The reminder shock did not recover fear memory in CORT-treated animals, suggesting that reconsolidation, not extinction, was affected. These results indicate that glucocorticoids and ß-adrenoceptors in the BLA jointly modulate fear memory reconsolidation and expression. Comprehending the neurobiology of stress and the impact of glucocorticoids on fear memory may lead to new treatments for stress and trauma-induced disorders such as PTSD.

A determination method for clenbuterol residue in pork based on optimal particle size gold colloid using SERS.

Clenbuterol is often used as a feed additive to increase the percentage of lean meat in livestock. Meat containing clenbuterol can cause many illnesses and even death for people. In this paper, the particle growth method was used to prepare gold colloids of different sizes, and the enhanced effectiveness of gold colloids of different sizes on clenbuterol in pork was investigated. The results showed that the gold colloid with the best enhanced effectiveness for clenbuterol had a particle size of approximately 90 nm. Second, a sample collection component was designed to detect clenbuterol from bottom to top, solving the problem of poor reproducibility of Surface-enhanced Raman scattering (SERS) detection caused by different droplet sizes and shapes. Then, the influence of different volumes of samples and concentrations of aggregating compounds on the enhanced effectiveness was optimized. The results showed that, based on the sample collection components designed in this article, 5 µL of enhanced substrate, 7.5 µL of clenbuterol and 3 µL of 1 mol/L mixed detection of NaCl solution had the best enhanced performance. Finally, 88 pork samples (0.5, 1, 1.5,…, 10, 12, 14 µg/g) with different concentrations were divided into correction sets and prediction sets in a ratio of 3:1. Unary linear regression models were established between the concentration of clenbuterol residue in the pork and the intensity of the bands at 390, 648, 1259, 1472, and 1601 cm-1. The results showed that the unary linear regression models at 390, 648, and 1259 cm-1 had lower root mean square errors than those at 1472 and 1601 cm-1. The intensity of the three bands and the concentration of clenbuterol residue in the pork were selected to establish a multiple linear regression model, and the concentration of clenbuterol residue in the pork was predicted. The results showed that the determination coefficients (R2) of the correction set and the prediction set were 0.99 and 0.99, respectively. The root mean square errors (RMSE) of the correction set and the prediction set were 0.169 and 0.184, respectively. The detection limit of clenbuterol in pork by this method is 42 ng/g, which can realize the crude screening of pork containing clenbuterol in the market.

Nanosensors: A smart remedy for early detection of clenbuterol contamination in food.

Veterinary drugs which are primarily meant for livestock treatment have now been categorised under potential food contaminant due to its unregulated usage and abuse. Their over usage by animal workers lead to production of contaminated animal-based food products which contain veterinary drug residues. These drugs are also misused as growth promoters to enhance the muscle to fat ratio in human body. This review highlights the misuse of such a veterinary drug; Clenbuterol. In this review, we have comprehensively discussed the usage of nanosensors to detect clenbuterol in food samples. Colorimetric, fluorescent, electrochemical, SERS and electrochemiluminescence are major categories of nanosensors that have been utilized for this purpose. The mechanism through which these nanosensors detect clenbuterol have been discussed in detail. The limit of detection and recovery percentage values of each nanosensor have been compared. This review will impart significant information on various nanosensors for clenbuterol detection in real samples.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.

OBJECTIVES: To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. METHODS: A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid-liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. RESULTS: R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5-500 ng/g. Accuracies ranged from -11.9-13.0% for R-(-)-clenbuterol and -10.2-13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6-5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. CONCLUSIONS: The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

"Blocking-effect" detection strategy of clenbuterol by molecularly imprinted electrochemiluminescence sensor based on multiple synergistic excitation of AgNW luminophores signal with highly active BNQDs@AuNFs nanoscale co-reaction accelerator.

A molecularly imprinted electrochemiluminescence sensor (MIECLS) is constructed to selectively detect clenbuterol (CLB) based on boron nitride quantum dots@gold nanoflowers/silver nanowires (BNQDs@AuNFs/AgNWs). The abundant amino and hydroxyl groups on the surface of the BNQDs generate an electrostatic self-assembly effect with the multi-tipped spatial structure of AuNFs, constituting a novel nanoscale co-reaction accelerator (NCRA) with high activity and large load capacity. An NCRA embedded in the network structure of the AgNW luminophores significantly promotes the reduction of peroxydisulfate (S2O82-) to sulfate anion radicals (SO4-•) through the catalysis of amino groups and boron radicals (B•) and the electron acceleration of AuNFs while also reducing the reaction distance between SO4-• and AgNWs-•, realizing the multiple synergistic amplification of the electrochemiluminescence (ECL) signal. Imprinted cavities in the molecularly imprinted polymers (MIPs) prepared by electropolymerization can generate a "blocking-effect" by recognizing CLB, realizing ECL signal quenching. Analytical results indicate that the established MIECLS detects CLB in a line concentration range of 0.5-50000 nM and detection limit of 0.00693 nM. The spiked recoveries are 85.90%-97.77%, with the relative standard deviations (RSD) under 5.1%, consistent with those of high-performance liquid chromatography (HPLC). This work demonstrates that an efficient NCRA can significantly enhance the output of the ECL signal in collaboration with the original luminophore, providing a new method to realize the ultra-detection of targeted substances by MIECLS.

Adverse events of clenbuterol among athletes: a systematic review of case reports and case series.

Clenbuterol is a potent beta-2 agonist widely misused by professional athletes and bodybuilders. Information on clenbuterol associated adverse events is present in case reports and case series, though it may not be readily available. This systematic review aimed to critically evaluate the evidence of adverse events associated with clenbuterol among athletes. The search strategy was in accordance with PRISMA guidelines. Databases such as PubMed, Science Direct, Scopus, and Google Scholar were searched from 1990 to October 2021 to find out the relevant case reports and case series. There were 23 included studies. Using a suitable scale, the included studies' methodological quality analysis was evaluated. In total, 24 athletes experienced adverse events. Oral ingestion of clenbuterol was the most preferred route among them. The daily administered dose of clenbuterol was ranging from 20 µg to 30 mg. Major adverse events experienced by athletes were supraventricular tachycardia, atrial fibrillation, hypotension, chest pain, myocardial injury, myocarditis, myocardial ischemia, myocardial infarction, cardiomyopathy, hepatomegaly, hyperglycemia, and death. The cardiac-related complications were the most commonly occurring adverse events. Clenbuterol is notorious to produce life-threatening adverse events including death. Lack of evidence regarding the performance-enhancing effects of clenbuterol combined with its serious toxicities questions the usefulness of this drug in athletes.

Opposing effects of ß-2 and ß-1 adrenergic receptor signaling on neuroinflammation and dopaminergic neuron survival in α-synuclein-mediated neurotoxicity.

BACKGROUND: Noradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson's disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, ß-adrenergic receptors' (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and ß-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood. METHODS: Two mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a ß-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after ß1-AR and ß2-AR agonist treatment in the hα-SYN virus-based model of PD. RESULTS: Consistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on ß-AR signaling since using a ß-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the ß-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a ß-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity. CONCLUSIONS: Our data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, ß2-AR specific agonists may have therapeutic benefit in PD.

Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2 µg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12 months at -20℃ and short-term stability for 7 days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

Quantification and enantiomeric distribution of clenbuterol in several bovine tissues using UHPLC-tandem mass spectrometry: Evaluation of a risk factor associated with meat contamination.

Clenbuterol (Clb) (4-amino-α-[(tert-butylamine) methyl]-3,5-dichlorobenzyl alcohol) is a sympathomimetic agent that exhibits ß2-agonist activity. It is applied as a bronchodilatory, tocolytic, and mucolytic agent and is authorized for clinical management in both human and veterinary therapeutics as a racemic mixture. However, its use is strictly prohibited in animals destined for food production in countries in the European Union and in the United States and Mexico, among many others. The R-(-) enantiomer in clenbuterol stimulates ß2-receptors, whereas the S-(+) enantiomer blocks the effect of ß1-receptors. The aims of this study were to develop a method for detecting and quantifying Clb and its enantiomeric distribution in several bovine tissues. The UHPLC-MS/MS method developed to quantify the target compound at trace levels in these tissues combines high sensitivity with good selectivity and short chromatographic run time. The tissue samples tested were found to contain racemic Clb in concentrations of 5-447 pg g-1 . The enantiomeric analysis of Clb showed that R-(-)-Clb is present at higher concentrations in some tissues, whereas S-(+)-Clb was detected in a ratio of 55/45 in the liver and heart tissues.

A lateral flow immunoassay based on chemisorbed probes in virtue of hydrogen bond receptors on the Bi2S3 NPs.

Improving detection sensitivity is still a major research emphasis for lateral flow immunoassay (LFIA). Increasing the binding efficiency and stability of the probe is an achievable and effective solution. In this work, we developed a highly sensitive lateral flow immunoassay for clenbuterol detection by using bismuth sulfide nanoparticle (Bi2S3) nanoparticles (NPs) as a novel marker. Here, Bi2S3 NPs can link with the antibody by hydrogen bonding to improve the performance of the probe, e.g., stability and sensitivity. Benefiting from the direct hydrogen bonding between Bi2S3 NPs and the monoclonal antibody (mAb), high sensitivity is obtained by the proposed LFIA with a lower visible detection limit of 0.1 ng mL-1 and a cut-off value of 4 ng·mL-1 for CLE detection, which is 5-fold and 7.5-fold improved than the conventional Au NPs based LFIA. In addition, the encouraging practical application results in milk, pork, and beef show that the bismuth sulfide nanoparticle has a great popularizing potential in the performance promotion of LFIAs for food safety monitoring.

Chemical staining enhanced Enzyme-linked immunosorbent assay for sensitive determination of Clenbuterol in food.

Exploring a novel strategy for strengthening the catalytic activity of enzyme facilitates the development of a sensitive enzyme-linked immunosorbent assay (ELISA). Herein, a chemical staining (CS) strategy was firstly discovered to possess the ability to directly improve the catalytic activity of horseradish peroxidase. Based on this discovery, coomassie brilliant blue was introduced into ELISA to establish a CS enhanced ELISA (CS-ELISA) to detect clenbuterol (CL) by simply staining monoclonal antibodies. Satisfactorily, the most important analytical parameters of CS-ELISA, including sensitivity (0.074 ng mL-1) and linear range (0.2-2 ng mL-1) were all improving 2-folds compared with conventional ELISA. Moreover, the CS-ELISA shows good applicability in the detection of CL in pork tenderloin samples. The proposed CS-ELISA shows various advantages, such as cost-effective, easily accessible, enhanced catalytic activity of enzyme, higher sensitivity, and broader linear range, providing a new insight into enhanced ELISA for food safety.

Rapid Detection of Clenbuterol Residues in Pork Using Enhanced Raman Spectroscopy.

Clenbuterol (CB) is a synthetic ß-receptor agonist which can be used to improve carcass leanness in swine, but its residues in pork also pose health risks. In this report, surface-enhanced Raman scattering (SERS) technology was used to achieve rapid detection and identification of clenbuterol hydrochloride (CB) residues. First, the effects of several different organic solvents on the extraction efficiency were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Then, SERS signals of clenbuterol in different solvents were compared, and it was found that clenbuterol had a better enhancement effect in an aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Next, enhancement effect was compared using different concentration of sodium chloride solution as the aggregating compound. Finally, pork samples with different clenbuterol content (1, 3, 5, 7, 9, and 10 µg/g) were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 mol/L of NaCl solution as aggregating compound and gold colloid as an enhanced substrate. Multiple scattering correction (MSC) and automatic Whittaker filter (AWF) were used for preprocessing, and the fluorescence background contained in the original Raman spectra was removed. A unary linear regression model was established between SERS intensity at 1472 cm-1 and clenbuterol content in pork samples. The model had a better linear relationship with a correlation coefficient R2 of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid screening of pork containing clenbuterol in the market.

Presence of ß2 -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses.

ß2 -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri-TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro-dose of 2.5 µg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml.

Effect of Clenbuterol on Muscle Activity During Exercise in Standardbred Horses.

Clenbuterol (ß2 agonist) is a commonly administered bronchodilator in race and performance horses. While long-term administration can alter exercise performance and muscle properties, little is known about its effects on these parameters following short-term administration. A single dose of clenbuterol (0.80 µg/kg) was expected to alter muscle activity of the extensor carpi radialis, semitendinosus, and longissimus dorsi during submaximal exercise. Eight mature Standardbred horses exercised for 2 minutes at 5 m/s on a high-speed treadmill following clenbuterol dosing (clenbuterol) or no dosing (control) in a crossover experimental design. Surface electromyography (sEMG) data were collected continuously from the muscles of interest and processed to determine average rectified value (ARV) and median frequency (MF) of the signal during peak muscle activation (100 ms period) during 15 strides. ARV data were log transformed. Data were analyzed with a mixed model ANOVA with fixed effects of period and treatment and a random effect of horse. No differences (P > .05) in amplitude (ARV) or frequency (MF) of the EMG signal were detected following clenbuterol administration. Thus, a one-time dose of clenbuterol had no statistically detectable effect on muscle activity during submaximal exercise. Further studies should be undertaken to confirm these results and examine the effects of long-term administration on muscle activity during exercise.

Clenbuterol attenuates immune reaction to lipopolysaccharide and its relationship to anhedonia in adolescents.

While inflammation has been implicated in psychopathology, relationships between immune-suppressing processes and psychiatric constructs remain elusive. This study sought to assess whether ß2-agonist clenbuterol (CBL) would attenuate immune activation in adolescents with mood and anxiety symptoms following ex vivo exposure of whole blood to lipopolysaccharide (LPS). Our focus on adolescents aimed to target a critical developmental period when psychiatric conditions often emerge and prior to chronicity effects. To capture a diverse range of immunologic and symptomatologic phenotypes, we included 97 psychotropic-medication free adolescents with mood and anxiety symptoms and 33 healthy controls. All participants had comprehensive evaluations and dimensional assessments of psychiatric symptoms. Fasting whole-blood samples were collected and stimulated with LPS in the presence and absence of CBL for 6 hours, then analyzed for 41 cytokines, chemokines, and hematopoietic growth factors. Comparison analyses used Bonferroni-corrected nonparametric tests. Levels of nine immune biomarkers-including IL-1RA, IL-1ß, IL-6, IP-10, MCP-1, MIP-1α, MIP-1ß, TGF-α, and TNF-α-were significantly reduced by CBL treatment compared to LPS alone. Exploratory factor analysis reduced 41 analytes into 5 immune factors in each experimental condition, and their relationships with psychiatric symptoms were examined as a secondary aim. CBL + LPS Factor 4-comprising EGF, PDGF-AA, PDGF-AB/BB, sCD40L, and GRO-significantly correlated with anticipatory and consummatory anhedonia, even after controlling for depression severity. This study supports the possible inhibitory effect of CBL on immune activation. Using a data-driven method, distinctive relationships between CBL-affected immune biomarkers and dimensional anhedonia were reported, further elucidating the role of ß2-agonism in adolescent affective symptomatology.