A Novel Method for the Production of an Autologous Anti-Inflammatory and Anti-Catabolic Product (Cytorich) from Human Blood: A Prospective Treatment for the COVID-19-Induced Cytokine Storm.

BACKGROUND Autologous blood-derived products can target specific inflammatory molecular pathways and have potentially beneficial therapeutic effects on inflammatory pathologies. The purpose of this study was to assess in vitro the anti-inflammatory and anti-catabolic potential of an autologous blood product as a possible treatment for COVID-19-induced cytokine storm. MATERIAL AND METHODS Blood samples from healthy donors and donors who had recovered from COVID-19 were incubated using different techniques and analyzed for the presence of anti-inflammatory, anti-catabolic, regenerative, pro-inflammatory, and procatabolic molecules. RESULTS The highest concentrations of therapeutic molecules for targeting inflammatory pathways were found in the blood that had been incubated for 24 h at 37°C, whereas a significant increase was observed after 6 h of incubation in blood from COVID-19-recovered donors. Beneficially, the 6-h incubation process did not downregulate anti-COVID-19 immunoglobulin G concentrations. Unfortunately, increases in matrix metalloproteinase 9, tumor necrosis factor alpha, and interleukin-1 were detected in the product after incubation; however, these increases could be blocked by adding citric acid, with no effect on the concentration of the target therapeutic molecules. Our data allow for safer and more effective future treatments. CONCLUSIONS An autologous blood-derived product containing anti-inflammatory and anti-catabolic molecules, which we term Cytorich, has a promising therapeutic role in the treatment of a virus-induced cytokine storm, including that associated with COVID-19.

Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program.

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

Development of an extraction method for anabolic androgenic steroids in dietary supplements and analysis by gas chromatography-mass spectrometry: Application for doping-control.

Several studies have highlighted that nutritional supplements may contain undeclared anabolic steroids that are banned by the International Olympic Committee/World Anti-Doping Agency. Any kind of abuse with these drugs is extremely dangerous because of their side effects. Thus, the control of food additives in order to protect the best consumer health and to limit fraudulent practices in the field of sports is essential. This paper describes a simple and effective qualitative gas chromatography-mass spectrometry (GC-MS) method to detect anabolic androgenic steroids (AAS): androsterone, nandrolene, dehydroepiandrosterone, 5ɑ-androstane-3ß, 17ß-diol, dihydrotestosterone, testosterone, methenolone acetate, methandienone, boldenone and fluoxymesterone, in food supplements. Methyltestosterone was used as internal standard. Target compounds were extracted with a mixture of N-pentane and di-ethylether (7.5:2.5, v/v). A good extraction recovery was obtained by our method for all the AAS (R > 88%). Crude extract was derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Separation was performed on a GC connected to quadrupole MS detector using a 5% phenylmethylsiloxane fused silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 µm). Helium was used as carrier gas with a flow rate of 0.3 µl min-1 (measured at 6.1 psi 190 °C). The MS was operated in electron ionization mode (70 eV) and in selected ion monitoring (SIM). The mass spectra of the standard compounds were acquired in full SCAN mode (50-700 m/z) by infusion of a reference solution at 50 µg/ml. Three higher diagnostic ions were monitored for each compound of interest. All AAS get separated with good peak shapes and resolution factor. The total analysis time by our optimised method was only 20 min. The developed method was validated according to Laboratories International Standard regulations for specificity, precision in both liquid and solid matrixes, and memory effect. The Tolerance Interval was judged true. The limit of detection was about 10 ng/g for solid samples and 10 ng/ml for liquid samples. The developed method was then applied to the research of steroids in nine Tunisian commercially dietary supplements using for each compound of interest SIM mode for screening then SCAN mode for confirmation. One of the monitoring samples was positive to methandienone not declared on the label. Our analytical method can be beneficial for AAS screening in dietary supplements.

Challenges in Modern Anti-Doping Analytical Science.

The challenges facing modern anti-doping analytical science are increasingly complex given the expansion of target drug substances, as the pharmaceutical industry introduces more novel therapeutic compounds and the internet offers designer drugs to improve performance. The technical challenges are manifold, including, for example, the need for advanced instrumentation for greater speed of analyses and increased sensitivity, specific techniques capable of distinguishing between endogenous and exogenous metabolites, or biological assays for the detection of peptide hormones or their markers, all of which require an important investment from the laboratories and recruitment of highly specialized scientific personnel. The consequences of introducing sophisticated and complex analytical procedures may result in the future in a change in the strategy applied by the Word Anti-Doping Agency in relation to the introduction and performance of new techniques by the network of accredited anti-doping laboratories.

An improved hollow fiber solvent-stir bar microextraction for the preconcentration of anabolic steroids in biological matrix with determination by gas chromatography-mass spectrometry.

In this paper, a convenient and self-assembled hollow fiber solvent-stir bar microextraction (HF-SSBME) device was developed, which could stir by itself. In the extraction process, the proposed device made the solvent "bar" not floating at the sample solution and exposing to air while organic solvents outside hollow fiber always wrapped with donor phase solvent, which reduced the vaporization of organic solvents. This design could improve the precisions and recoveries of experiments. For evaluating the device, seven anabolic steroids (prasterone, 5α-androstane-3α, 17ß-diol, methandriol, 19-norandrostenediol, androstenediol, methyltestosterone and methandienone) were used as model analytes and extraction conditions such as type and volume of organic solvents, agitation speed, extraction time, extraction temperature and salt addition were studied in detail. Under the optimum conditions (15 µL toluene, 40 °C, stirring at 750 rpm for 30 min with 1.5 g sodium chloride addition in 20.0 mL donor phase), the linear ranges of anabolic steroids were 0.25-200 ng mL(-1) with gas chromatography-mass spectrometry. The limits of detection were lower than 0.10 ng mL(-1). The recoveries and precisions in spiked urine and hair samples were between 73.97-93.56% and 2.18-4.47% (n=5). HF-SSBME method combined the intrinsical merits of hollow fiber with the superiority of the proposed self-stirring device which can be developed to two-phase, three-phase and in situ derivatization modes with wide prospect of application. Besides, the pedestal of this proposed device can be converted to fix stir bar in stir bar sorptive extraction (SBSE) method.

The potential of bovine vaginal smear for biomarker development to trace the misuse of anabolic agents.

In the European Union the use of anabolic hormones in meat production is forbidden since 1988 and this ban of anabolic agents in animal production is strictly controlled. New hormone cocktails passing the detection systems are attractive for the practice and so new approaches to discover their illegal use have to be developed steadily. Verifying physiological effects caused by anabolic steroids will be a new way to develop potential monitoring systems. One promising matrix in female animals will be vaginal smear containing vaginal epithelial cells, because the vaginal epithelium is a primary steroid hormone responsive organ. In this study we quantified the gene expression in vaginal smear of sexually mature cattle in order to observe physiological effects. Further we aimed to establish a new screening method by testing the effect of a combination of certain anabolic steroid hormones on physiological regulations of mRNA expression of selected genes. In an animal trial Nguni heifers were treated with the anabolic combination trenbolone acetate plus estradiol. Vaginal smear samples were taken at 4 different time points. Gene expression of 27 candidate genes, selected by screening the actual literature for steroidal effects on vaginal epithelial cells, were estimated using quantitative real-time RT-PCR. There were different expression changes observed at different time points. It could be shown that the applied anabolic combination significantly influenced the expression of the steroid receptor ERα, the keratinization factor CK8, the proinflammatory interleukins IL-1α and IL-1ß, the growth factors FGF7, EGF, EGFR, IGF-1R, TGFα and LTF, the oncogen c-jun and other factors like actinß and ubiquitin 3. Using biostatistical tools like principal components analysis or hierarchical cluster analysis, the potential to develop a gene expression pattern for targeting the illegal use of growth promoters could be demonstrated.

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC(50) of 0.79 ngmL(-1) for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11beta-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Detection of the arylpropionamide-derived selective androgen receptor modulator (SARM) S-4 (Andarine) in a black-market product.

Non-steroidal and tissue-selective anabolic agents such as selective androgen receptor modulators (SARMs) represent a promising class of therapeutics for the treatment of various diseases such as sarcopenia or cancer cachexia. Advanced compounds of SARMs are based on an arylpropionamide-derived structure and leading drug candidates have successfully completed phase-II-clinical trials. Although none of these therapeutics have been approved, their performance-enhancing qualities and the black-market availability of these products makes them a viable target for misuse in the athletic community. In 2008, SARMs were added to the Prohibited List established by the World Anti-Doping Agency (WADA). That SARMs are the subject of misuse even without clinical approval was proved for the first time by the detection of the drug candidate Andarine (also referred to as S-4, S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide), advertised, sold and supplied via the Internet. The oily liquids, declared as green tea extracts and face moisturizer, were assayed using state-of-the-art analytical procedures and S-4 was found at concentrations of approximately 150 mg/mL. The authenticity of the product was demonstrated in comparison to reference material by liquid chromatography, high resolution/high accuracy (tandem) mass spectrometry using positive and negative electrospray ionization, and comparison to reference material. Moreover, an impurity resulting from poor product purification was detected, accounting for approximately 10% of S-4. This consisted of 2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-3-(4-nitro-3-trifluoromethyl-phenylamino)-propionamide. The ease of purchasing non-approved drug candidates that could potentially increase athletic performance demonstrates the need to operate proactively in the continued fight against doping. The early inclusion of emerging drugs into routine sports drug testing procedures is a key element of preventive doping research, limiting the options for cheating athletes who aim to undermine the doping control system.

Automated sample preparation and gas chromatographic-mass spectrometric analysis of urinary androgenic anabolic steroids.

This paper presents an automated method for extracting anabolic agents from urine samples for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 microl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.

[Synthetic anabolic androgens in the fecal material of cattle: radioimmunological determination after purification by high performance liquid chromatography]. / Androgènes anabolisants de synthèse dans les matières fécales des bovins: dosage radio-immunologique après purification par chromatographie liquide à haute performance.

In answer to the mandatory control of the illegal use of anabolizing agents in meat-producing animals imposed by the EEC in farms, a method of analysis of faeces involving high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) has been described. Four HPLC fractions were collected and submitted to corresponding RIA: 17 beta- and 17 alpha-trenbolone, 17 beta-nortestosterone, 17 alpha-nortestosterone and 17 alpha-methyltestosterone. The mean extraction and purification yield was estimated at 44 +/- 7% using tritiated 17 alpha-methyltestosterone as internal standard. Detection limits of the 3 hormones were estimated at 0.2-0.3 ng/g of faeces. About 50 samples can be analysed per week by this method.