LIPOSOMES: DRUG DELIVERY SYSTEM OR POSSIBLE DOPING AGENT?

The use of liposomes as drug delivery vehicle for treatment of various diseases is well known in medical field but its possible role as a masking agent in sports came into light when Liposom Forte® was found stored together with banned and non-banned drugs during investigations carried out by Italian legal authorities and recent availability of IGF-1 Liposomal Spray on internet. Role of liposomes as masking agent for anabolic steroids in the field of doping has been investigated by Botre et al. The aim of the present work was to study the effect of different parameters like temperature, pH, charge, time, concentration etc. on the interaction of liposomes and doping agents and to identify a possible marker for detection of their abuse in sports. The results showed that out of variety of doping agents, the direct addition of liposomes to urine samples containing anabolic steroids shows strong tendency to interact with the liposomes which results in the reduced concentration of the compound in the sample. However, there was no significant effect of temperature and incubation time on the interaction of liposome and doping agents while other parameters such as charge and concentration of liposome affect the interaction capacity. Keywords: WADA, Liposomes, DopingÂ

URINE EXCRETION STUDY OF TAMOXIFEN METABOLITE, 3-HYDROXY-4-METHOHY TAMOXIFEN BY GC-MS

Background; Tamoxifen, is a first-generation selective estrogen receptor modulator which  is widely used for adjuvant breast cancer therapy. Tamoxifen metabolite completely bind to estrogen receptor on tumors as well as other tissues and target a nuclear complex that decreases DNA synthesis and inhibits estrogen effects. But these Selective Estrogen Receptor Modulators (SERMs)  are classified as Prohibited substances according to the list of forbidden substances in sports by the World Anti-Doping Agency(WADA) In sports male athletes use tamoxifen in fighting against increase of estrogen level in the body, breast swelling , gynecomastia and fat forming a pear-silhouette very successfully. Aim; In the present study  Tamoxifen was investigated carefully by administering to a Healthy volunteer . Material And Method; Urine samples are collected at different hours  from 1st day to 5th day and analyzed by GC-MS. Result; It was  found that Tamoxifen is rapidly metabolized in to their metabolite 3-hydrohy-4-methoxy Tamoxifen. Urinary extracts were analyzed by Gas Chromatography-Mass spectrometric Detector by using Selective ion modulator(SIM) and targeted GC-MS techniques with accurate mass. By the GC-MS study it is observed that 3-hydroxy-4-methoxy Tamoxifen metabolite gives two neutral loss m/z value at 58 & 72,  but m/z value 58 shows more abundance than 72m/z value, hence it was selected as the marker m/z  value to check the presence of metabolite in urine. Conclusion; After oral administration, Tamoxifen metabolite can be detected in to their maximum period up to 5 days.Â

Untargeted Metabolomics Profiling Reveals Perturbations in Arginine-NO Metabolism in Middle Eastern Patients with Coronary Heart Disease

Coronary heart disease (CHD) is a major cause of death in Middle Eastern (ME) populations, with current studies of the metabolic fingerprints of CHD lacking in diversity. Identification of specific biomarkers to uncover potential mechanisms for developing predictive models and targeted therapies for CHD is urgently needed for the least-studied ME populations. A case-control study was carried out in a cohort of 1001 CHD patients and 2999 controls. Untargeted metabolomics was used, generating 1159 metabolites. Univariate and pathway enrichment analyses were performed to understand functional changes in CHD. A metabolite risk score (MRS) was developed to assess the predictive performance of CHD using multivariate analysis and machine learning. A total of 511 metabolites were significantly different between the CHD patients and the controls (FDR p < 0.05). The enriched pathways (FDR p < 10−300) included D-arginine and D-ornithine metabolism, glycolysis, oxidation and degradation of branched chain fatty acids, and sphingolipid metabolism. MRS showed good discriminative power between the CHD cases and the controls (AUC = 0.99). In this first study in the Middle East, known and novel circulating metabolites and metabolic pathways associated with CHD were identified. A small panel of metabolites can efficiently discriminate CHD cases and controls and therefore can be used as a diagnostic/predictive tool

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC–MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation

Ultra-Fast Retroactive Processing by MetAlign of Liquid-Chromatography High-Resolution Full-Scan Orbitrap Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program

Rationale: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. Methods: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). Results: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. Conclusions: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.</p

A simple and rapid ESI-LC-MS/MS method for simultaneous screening of doping agents in urine samples

Objective: The use of performance enhancing substances is banned in sports by the World Anti-Doping Agency (WADA). Though most prohibited substances can be detected by GC/MS, inclusion of corticosteroids and designer drugs has made it essential to detect these critical doping agents on LC/MS/MS due to their better separation and detection. Materials and Methods: A common extraction procedure for the isolation of acidic, basic and neutral drugs from urine samples was developed. A total of 28 doping drugs were analyzed on API 3200 Triple quadrupole mass spectrometer using C18 column in atmospheric pressure electrospray ionization. The mobile phase composition was a mixture of 1% formic acid and acetonitrile with gradient time period. Results: The method developed was very sensitive for detection of 28 doping agents. The linearity was performed for each drug and the total recovery percentage ranged from 57 to 114. Limit of detection is found to be 0.5 ng/ml for carboxy finasteride and 1-5 ng/ml for other drugs. The method was successfully used to detect positive urine samples of 3-OH-stanozolol, methyl phenidate, mesocarb, clomiphene metabolite and carboxy finasteride. Conclusion: The method developed based on controlled pH extraction method and HPLC-mass spectrometry analysis allowed better identification and confirmation of glucocorticosteroids and a few other drugs in different categories. The validated method has been used successfully for testing of 1000 In-competition samples. The method helped in detection of chemically and pharmacologically different banned drugs in urine in a single short run at a minimum required performance limit set by WADA

Analysis of glucocorticosteroids by atmospheric pressure chemical ionization-liquid chromatography mass spectrometry (APCI-C/MS/MS)

Objective: To develop a rapid liquid chromatography / mass spectrometry / mass spectrometry (LC/MS/MS) method for testing of glucocorticosteroids, which are banned in sports by the World Anti-doping Agency from January 1 st 2004. Materials and Methods : A total of 14 glucocorticosteroids were analyzed on LC/MS/MS using an Inertsil ® ODS-3 (3.0 µm, 50 mm ´ 4.6 mm i.d.) C-18 column in atmospheric pressure chemical ionization mode (positive ionization) with a mobile phase consisting of ammonium acetate and acetonitrile. The analytical equipment used was Aglient 1100 HPLC and API-3200 Triple quadrupole mass spectrometer. Results : All glucocorticosteroids could be detected within 8 minutes. The limit of detection of all glucocorticosteroids by this screening method was 1 ng/ml. The recovery percentage at 25 and 50 ng/ml concentrations ranged from 54% (Prednisone) to 144% (Methylprednisolone).The validated method has been used successfully for testing of 500 in-competition samples. Excretion study samples of budesonide, methyl prednisolone and prednisone were analyzed by this method and the parent drugs as well as metabolites could be detected. However, further work is in progress to combine this procedure with another LC/MS/MS procedure in the ESI mode (positive ionization) being used for few anabolic steroids and heat-labile stimulants. This would help in screening of all corticosteroids, few anabolic agents and stimulants with just one injection, thus saving time and effort

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35&ndash;36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p &lt; 0.05 and met &ge; 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT

20-hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner

Background: 20-hydroxyecdysone is an ecdysteroid which is abundant in plants and insects and has anabolic potentials in mammals. It was recently shown to have affinity for estrogen ER-β receptor, which could potentially make it vasodilatory. Yet this possibility has not been previously investigated. Such an activity in muscle arterioles could have huge implications for muscle blood flow and performance. Hypothesis/Purpose: We hypothesized that 20-hydroxyecdysone would dilate muscle arterioles by activating estrogen ER-β receptors. To test this, we investigated its vasodilatory properties in ovine muscle arterioles and further explored the mechanisms in human tissues and cells. Study Design: The study was carried out experimentally, employing functional recording of arteriolar reactivity in intact ovine muscle arterioles and gene and protein expression analysis in human tissues and cells. Methods: Direct effects of the compound on arteriolar tone were assessed by wire myography in abdominal muscle and mesenteric arterioles isolated from samples obtained from male sheep. The roles of endothelial nitric oxide synthase (NOS3), cyclooxygenase (COX) and estrogen ER-β receptor (ER-β), and their effects were determined with specific blockers. The NOS3 mRNA and protein expressions were analyzed in human coronary artery endothelial cells (HCAECs) and humanized liver of uPA+/+-SCID mice, before and after 20-hydroxyecdysone administration. Results: Comparable dose-dependent relaxations were recorded for 20-hydroxyecdysone in both muscle and mesenteric arterioles with maximum relaxations of 46.94 ± 5.84% and 56.88 ± 7.04% respectively, which were not statistically different. Similar relaxation was recorded for β-estradiol in both arterioles. NOS inhibition with 100 µM L-NAME attenuated the relaxation to 20-hydroxyecdysone (p < 0.001) and β-estradiol (p < 0.001) in muscle arterioles. Neither COX inhibition with 10 µM indomethacin nor ER blockade with 1 µM PHTPP or 1 µM ICI182780 had any noticeable effect on 20-hydroxyecdysone relaxation in these arterioles. Transcriptome analysis revealed elevated NOS3 gene in the humanized liver of 20-hydroxyecdysone-treated mice, and, elevation of both NOS3 mRNA and protein in HCAECs treated with 20-hydroxyecdysone. Conclusion: The data suggest that 20-hydroxyecdysone has a nitric oxide-dependent, but ERβ-independent, vasodilatory property in muscle arterioles. The benefits to muscle blood flow would however be dependent on the impact of its effects on other vascular beds

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35&ndash;36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p &lt; 0.05 and met &ge; 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT