Influence of mobile phase composition on the analytical sensitivity of LC-ESI-MS/MS for the concurrent analysis of bisphenols, parabens, chlorophenols, benzophenones, and alkylphenols.

Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a "dilute and shoot" approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.

Crystal structure of the Pseudomonas aeruginosa PA0423 protein and its functional implication in antibiotic sequestration.

Pseudomonas aeruginosa is a widely found opportunistic pathogen. The emergence of multidrug-resistant strains and persistent chronic infections have increased. The protein encoded by the pa0423 gene in P. aeruginosa is proposed to be critical for pathogenesis and could be a virulence-promoting protease or a bacterial lipocalin that binds a lipid-like antibiotic for drug resistance. Although two functions of proteolysis and antibiotic resistance are mutually related to bacterial survival in the host, it is very unusual for a single-domain protein to target unrelated ligand molecules such as protein substrates and lipid-like antibiotics. To clearly address the biological role of the PA0423 protein, we performed structural and biochemical studies. We found that PA0423 adopts a single-domain ß-barrel structure and belongs to the lipocalin family. The PA0423 structure houses an internal tubular cavity, which accommodates a ubiquinone-8 molecule. Furthermore, we reveal that PA0423 can directly interact with the polymyxin B antibiotic using the internal cavity, suggesting that PA0423 has a physiological function in the antibiotic resistance of P. aeruginosa.

Ionization of Gas-Phase Polycyclic Aromatic Hydrocarbons in Electrospray Ionization Coupled with Gas Chromatography.

Herein, gas-phase polycyclic aromatic hydrocarbons (PAHs) as nonpolar compounds were ionized to protonated molecular ions [M + H]+ without radical cations and simultaneously analyzed using gas chromatography (GC)/electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). The ionization profile, dissociation, and sensitivity were first investigated to understand the significant behavior of gas-phase PAHs under ESI. The formation of protonated molecular ions of PAHs was distinguished according to the analyte phase and ESI spray solvents. The protonated PAHs exhibited characteristic dissociations, such as H-loss, H2-loss, and acetylene-loss, via competition of internal energy. In addition, GC/ESI-MS/MS resulted in relatively lower concentration levels (better sensitivity) for the limits-of-detection (LODs) of PAHs than liquid chromatography (LC)/ESI-MS/MS, and it seems to result from the characteristic ionization mechanism of the gas-phase analyte under ESI. Furthermore, the LODs of gas-phase PAHs depended on molecular weight and proton affinity (PA). Consequently, we demonstrated the relationship among the analyte phases, sensitivities, and structural characteristics (molecular weight and PA) under ESI. The gas-phase PAHs provided enhanced protonation efficiency and sensitivity using GC/ESI-MS/MS, as their molecular weight and PA increased. Based on these results, we offered important information regarding the behavior of gas-phase analytes under ESI. Therefore, the present GC/ESI-MS/MS method has potential as an alternative method for simultaneous analysis of PAHs.

Development of a multi-functional concurrent assay using weak cation-exchange solid-phase extraction (WCX-SPE) and reconstitution with a diluted sample aliquot for anti-doping analysis.

RATIONALE: In addition to the development of adequate screening methods for multiple compounds, the World Anti-Doping Agency (WADA) requires anti-doping laboratories to analyze prohibited substances and their metabolites from various classes. This task presents a difficult challenge for all agencies and interests involved in the field of doping control. METHODS: A screening method is reported in which hybrid sample preparation was performed using a combination of weak cation-exchange solid-phase extraction (WCX-SPE) and the 'Dilute and Shoot' strategy in order to take advantage of both the methodologies. Target substances were extracted using a WCX cartridge and reconstituted with a diluted sample aliquot that included 20% of an untreated urine sample. The target substances were further analyzed by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS). RESULTS: The SPE procedure was optimized using a cartridge-washing step, elution conditions, and elution volume. The cartridge-washing step, which was performed using 10% methanol, improved the overall recovery of target substances. Since the recovery was observed to vary according to the pH of the eluting solution, we applied an elution step using both an acid and a basic organic solvent to achieve complementary recovery. Reconstitution of the diluted aliquot sample was performed to recover the polar substances. CONCLUSIONS: The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.

NLRP3 Inflammasome Contributes to Lipopolysaccharide-induced Depressive-Like Behaviors via Indoleamine 2,3-dioxygenase Induction.

Background: Inflammation may play a significant role in the pathogenesis of depression, although the molecular target for the treatment of inflammation-mediated depressive symptoms remains to be elucidated. Recent studies have implicated the NLRP3 inflammasome in various psychiatric disorders, including depression. However, the underlying mechanism by which NLRP3 inflammasome activation mediates the progression of depressive-like behaviors remains poorly understood. Methods: We examined whether NLRP3 deficiency influenced depressive-like behaviors and cerebral inflammation following systemic administration of lipopolysaccharide in mice. To further assess the contribution of the NLRP3 inflammasome to the progression of depression, we evaluated the effects of NLRP3 signaling on levels of indoleamine 2,3-dioxygenase. Results: Nlrp3-deficient mice exhibited significant attenuation of depressive-like behaviors and cerebral caspase-1 activation in a lipopolysaccharide-induced model of depression. Treatment with the antidepressant amitriptyline failed to block NLRP3-dependent activation of caspase-1, but inhibited lipopolysaccharide-promoted production of interleukin-1ß mRNA via suppressing NF-κB signaling in mouse mixed glial cultures. Interestingly, lipopolysaccharide administration produced NLRP3-dependent increases in indoleamine 2,3-dioxygenase expression and activity of mouse brain. Furthermore, inflammasome-activating stimulations, but not treatment with the inflammasome product interleukin-1ß, triggered indoleamine 2,3-dioxygenase mRNA induction in mixed glial cells. Conclusions: Our data indicate that the NLRP3 inflammasome is significantly implicated in the progression of systemic inflammation-induced depression. NLRP3-dependent caspase-1 activation produced significant increases in indoleamine 2,3-dioxygenase levels, which may play a significant role in lipopolysaccharide-induced depression. Collectively, our findings suggest that indoleamine 2,3-dioxygenase is a potential downstream mediator of the NLRP3 inflammasome in inflammation-mediated depressive-like behaviors.

Dependency of Experimental Autoimmune Encephalomyelitis Induction on MOG35-55 Properties Modulating Matrix Metalloproteinase-9 and Interleukin-6.

Experimental autoimmune encephalomyelitis (EAE) is commonly induced with myelin oligodendrocyte glycoprotein (MOG)35-55; occasionally, EAE is not well induced despite MOG35-55 immunization. To confirm that EAE induction varies with difference in MOG35-55 properties, we compared three MOG35-55 from different commercial sources, which are MOG-A, MOG-B, and MOG-C. The peptides induced EAE disease with 100, 40, and 20 % incidence, respectively. Compared with others, MOG-A showed higher peptide purity (99.2 %) and content (92.2 %) and presented a sheet shape with additional sodium and chloride chemical elements. In MOG-A-treated group, MMP-9 activity and IL-6 levels were considerably higher than the other groups in CNS tissues, and significantly increased VCAM-1, IFN-γ, and decreased IL-4 were also shown compared to MOG-B- and/or MOG-C-treated group. In conclusion, the immunological and toxicological changes by the difference in MOG35-55 properties modulate EAE induction, and MOG35-55 which affects MMP-9 activity and IL-6 levels may be the most effective EAE-inducing antigen. This study can be potentially applied by researchers using MOG35-55 peptide and manufacturers for MOG35-55 synthesis.

Evaluation of exposure to cyanogenic glycosides and potential hydrogen cyanide release in commercially available foods among the Korean population.

The release of hydrogen cyanide (HCN) after food ingestion can pose a serious health risk to consumers. This study aimed to simultaneously quantify four cyanogenic glycosides (lotaustralin, prunasin, taxiphyllin, and dhurrin) using liquid chromatography-tandem mass spectrometry. The analysis scope extended beyond agricultural products to various consumer foods to estimate dietary exposure to cyanogenic glycosides and assess its risk levels. The major exposure sources are cassava chips (lotaustralin), apples (seeds) (prunasin and dhurrin), and Prunus mume axis (taxiphyllin). In addition to quantifying specific cyanogenic glycosides, this study proposed the development of a preliminary risk assessment framework based on the dietary exposure assessment and the calculation of theoretical levels of HCN derived from cyanogenic glycoside concentrations. In the absence of established guidelines for the permissible intake of foods containing cyanogenic glycosides, this study provides initial guidance for assessing the risks associated with a range of commonly consumed foods.

Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro.

BACKGROUND: TB-500 (Ac-LKKTETQ), derived from the active site of thymosin ß4 (Tß4), has various biological functions in its unacetylated form, LKKTETQ. These functions include actin binding, dermal wound healing, angiogenesis, and skin repair. The biological effects of TB-500, however, have not been documented. And the analysis of TB-500 and its metabolites have been neither simultaneously quantified nor structurally identified using synthesized authentic standards. METHODS: This study was aimed to investigating simultaneous analytical methods of TB-500 and its metabolites in in-vitro and urine samples by using UHPLC-Q-Exactive orbitrap MS, and to comparing the biological activity of its metabolites with the parent TB-500. The metabolism of TB-500 was investigated in human serum, various in-vitro enzyme systems, and urine samples from rats treated with TB-500, and their biological activities measured by cytotoxicity and wound healing experiments were also evaluated in fibroblasts. RESULTS: The simultaneous analytical method for TB-500 and its metabolites was developed and validated. The study found that Ac-LK was the primary metabolite with the highest concentration in rats at 0-6 h intervals. Also, the metabolite Ac-LKK was a long-term metabolite of TB-500 detected up to 72 hr. No cytotoxicity of the parent and its metabolites was found. Ac-LKKTE only showed a significant wound healing activity compared to the control. CONCLUSION: The study provides a valuable tool for quantifying TB-500 and its metabolites, contributing to the understanding of metabolism and potential therapeutic applications. Our results also suggest that the previously reported wound-healing activity of TB-500 in literature may be due to its metabolite Ac-LKKTE rather than the parent form.

Development of an Ephedrine In-House Matrix Reference Material and Its Application to Doping Analysis.

Biomatrix-based reference materials (RMs) improve the quality of laboratory test results by better representing actual samples. However, a matrix RM of ephedrine (EP) for threshold substances that require accurate analysis results has not yet been developed. Therefore, this study aimed to develop an in-house matrix RM for EP and subsequently apply it to analytical procedures. During the development of the in-house matrix EP RM, the system underwent homogeneity and stability studies. Additionally, it was subjected to interlaboratory comparison study in 11 laboratories, including 10 World Anti-Doping Agency (WADA)-accredited laboratories and our laboratory. Stability testing revealed no significant changes in the RM characteristics. For homogeneity, 10 random batches out of 200 were analyzed to confirm the uniformity within and between bottles. These results, combined with data from 11 laboratories, ensured retroactive validation. The traceability value of the in-house matrix EP RM was assigned as 9.83 ± 0.57 µg/mL (k = 2) by interlaboratory comparison studies and traceable uncertain evaluation. The feasibility of this method as a single calibration standard was confirmed in two laboratories. This substance is reliable and consistent for quality control during EP quantification, ensuring accurate and trustworthy outcomes. Consequently, this study establishes a framework and guidelines for producing in-house matrix RMs and serves as a reference for generating similar matrix RMs in other contexts.

Simple visualization method for the c.577del of erythropoietin variant: CRISPR/dCas9-based single nucleotide polymorphism diagnosis.

One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild-type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti-Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti-doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant-specific single-guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native-PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild-type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3 h using only 3 µl of the whole blood.

CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection.

A genetic approach targeted toward improving athletic performance is called gene doping and is prohibited by the World Anti-Doping Agency. Currently, the clustered regularly interspaced short palindromic repeats-associated protein (Cas)-related assays have been utilized to detect genetic deficiencies or mutations. Among the Cas proteins, deadCas9 (dCas9), a nuclease-deficient mutant of Cas9, acts as a DNA binding protein with a target-specific single guide RNA. On the basis of the principles, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene detection. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin-polyHRP that enables rapid signal amplification. For efficient biotin labeling via maleimide-thiol chemistry, two cysteine residues of dCas9 were structurally validated, and the Cys574 residue was identified as an essential labeling site. As a result, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (7.41 × 105 copies) and up to 10 nM (6.07 × 1011 copies) in a whole blood sample within 1 h with HiGDA. Assuming an exogenous gene transfer scenario, we added a direct blood amplification step to establish a rapid analytical procedure while detecting target genes with high sensitivity. Finally, we detected the exogenous human erythropoietin gene at concentrations as low as 2.5 copies within 90 min in 5 µL of the blood sample. Herein, we propose that HiGDA is a very fast, highly sensitive, and practical detection method for actual doping field in the future.

Metabolites identification of anabolic steroid bolasterone in vitro and in rats by high resolution liquid chromatography mass spectrometry.

Bolasterone (7α,17α-dimethyltestosterone) and anabolic androgenic steroids are included in the World Anti-Doping Agency's Prohibited list of substances. This study aimed to evaluate the metabolism of bolasterone through in vitro experiments using rat liver microsomes and in vivo experiments using rat urine after oral administration. Urine samples were collected over a 168-h period. Bolasterone and its metabolites were detected by liquid chromatography coupled with a Q-Exactive Obitrap mass spectrometry (LC-HRMS). Ultimately 16 hydroxylated metabolites (M1-M16), one metabolite from the reduction of the 3-keto function and 4-ene (M17), and one glucuronic acid conjugated metabolite (M18) were detected. Metabolites M17 and M18 were confirmed by comparison with available reference or authentic standards. Metabolic modifications in the structure of the parent bolasterone result in different fragmentation patterns. Based on the sensitivity of the HRMS data, characteristic ions such as m/z 121.064 (C8 H9 O) generated from ring A of the mono-hydroxylated metabolites and 121.101 (C9 H13 ) generated from ring D of the di-hydroxylated metabolites were observed that helped differentiate between the obtained metabolites. The structures of fragment ions were tentatively proposed based on their fragmentation pathways, where the significant ions were correlated to the possible structural fragments. In conclusion, new metabolites of bolasterone were detected and characterized by the use of the full-scan and dd-MS/MS using LC-HRMS, and this data can be useful for providing metabolite information for the interpretation of mass spectra of anabolic bolasterone analogues for doping screening tests.

Untargeted Metabolomics Analysis Reveals Toxicity Based on the Sex and Sexual Maturity of Single Low-Dose DEHP Exposure.

Di-(2-Ethylhexyl) phthalate (DEHP) is a prevalent environmental endocrine disruptor that affects homeostasis, reproduction, and developmental processes. The effects of DEHP have been shown to differ based on sex and sexual maturity. This study examines the metabolic profiles of mature adult rats from both sexes, aged 10 weeks, and adolescent female rats, aged 6 weeks, following a single 5 mg/kg of body weight DEHP oral administration. An untargeted metabolomic analysis was conducted on urine samples collected at multiple times to discern potential sex- and maturity-specific DEHP toxicities. Various multivariate statistical analyses were employed to identify the relevant metabolites. The findings revealed disruptions to the steroid hormone and primary bile acid biosynthesis. Notably, DEHP exposure increased hyocholic, muricholic, and ketodeoxycholic acids in male rats. Moreover, DEHP exposure was linked to heart, liver, and kidney damage, as indicated by increased plasma GOT1 levels when compared to the levels before DEHP exposure. This study provides detailed insights into the unique mechanisms triggered by DEHP exposure concerning sex and sexual maturity, emphasizing significant distinctions in lipid metabolic profiles across the different groups. This study results deepens our understanding of the health risks linked to DEHP, informing future risk assessments and policy decisions.

Production and engineering of nanobody-based quenchbody sensors for detecting recombinant human growth hormone and its isoforms.

Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti-Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence-incorporated antibody sensor "Quenchbody (Q-body)" that activates upon antigen binding. Camelid-derived nanobodies were used to produce stable Q-bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q-bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis-tag, and Q-bodies were obtained using a maleimide-thiol reaction between the N-terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58-fold to 3.04-fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA-labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520-labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q-bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti-doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.

Evaluation and Monitoring of the Natural Toxin Ptaquiloside in Bracken Fern, Meat, and Dairy Products.

Ptaquiloside, a naturally occurring cancer-causing substance in bracken fern, has been detected in the meat and milk of cows fed a diet containing bracken fern. A rapid and sensitive method for the quantitative analysis of ptaquiloside in bracken fern, meat, and dairy products was developed using the QuEChERS method and liquid chromatography-tandem mass spectrometry. The method was validated according to the Association of Official Analytical Chemists guidelines and met the criteria. A single matrix-matched calibration method with bracken fern has been proposed, which is a novel strategy that uses one calibration for multiple matrices. The calibration curve ranged from 0.1 to 50 µg/kg and showed good linearity (r2 > 0.99). The limits of detection and quantification were 0.03 and 0.09 µg/kg, respectively. The intraday and interday accuracies were 83.5-98.5%, and the precision was <9.0%. This method was used for the monitoring and exposure assessment of ptaquiloside in all routes of exposure. A total of 0.1 µg/kg of ptaquiloside was detected in free-range beef, and the daily dietary exposure of South Koreans to ptaquiloside was estimated at up to 3.0 × 10-5 µg/kg b.w./day. The significance of this study is to evaluate commercially available products in which ptaquiloside may be present, to monitor consumer safety.

Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC-HRMS.

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood's capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.

Application of Skyline software for detecting prohibited substances in doping control analysis.

As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.

Detection and quantification of the metabolite Ac-Tß1-14 in in vitro experiments and urine of rats treated with Ac-Tß4: A potential biomarker of Ac-Tß4 for doping tests.

Thymosin ß4 (Tß4) was reported to exert various beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti-Doping Agency. However, no metabolism studies of Tß4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tß31-43 , Ac-Tß17-43 , Ac-Tß1-11 , Ac-Tß1-14 , Ac-Tß1-15 , and Ac-Tß1-17 ) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tß4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tß4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac-Tß1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac-Tß1-14 were 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r2 = 0.9998). As a result, among the six metabolites above, Ac-Tß1-14 , as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tß4, and the urine of rats intraperitoneally treated with 20-mg/kg Tß4. And the metabolite Ac-Tß1-14 was quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac-Tß1-14 was not detected in non-treated control groups, including human blank urine. These results suggest that Ac-Tß1-14 in urine is a potential biomarker for screening the parent Tß4 in doping tests.

Minor red blood cell antigen phenotyping of athletes sampled in international competitions.

Blood transfusion is performed by cheating athletes to rapidly increase oxygen delivery to exercise muscles and enhance their performance. This method is banned by the World Anti-doping Agency (WADA). Heterologous or allogenic blood transfusion happens when blood from a different person is transfused. The method used to detect this type of doping is based on flow cytometry, by identifying variations in blood group minor antigens present on the red blood cells' surface. Transfusion practices have regained interest since the introduction of human recombinant erythropoietin detection method. It has been reported that the number of occurrences of two athletes sharing an identical phenotype in the same sport was five times higher than the theoretical populational probability. The present work describes the prevalence of 10 erythrocytes surface antigens in a population of 261 athletes from all five continents. The matching phenotype per sport is also described.

New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis.

The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping.