Androgenic anabolic steroids (AASs) are synthetic derivatives of testosterone with a common structure containing cyclopentanoperhydrophenanthrene nucleus. Their use enhances the muscle building ...capacity and is beneficial during performance. The AASs are one of the most abused group of substances in horse doping. Liquid chromatography tandem mass spectrometry ($LC/MS^n$) has been successfully applied to the detection of anabolic steroids in biological samples. However, the saturated hydroxysteroids viz: nandrolone, $5{\alpha}-estrane-3{\beta}$, $17{\alpha}-diol$ exhibit lower detection responses in electrospray ionisation (ESI) because of their poor ionisation efficiency. To overcome this limitation pre-column chemical derivatization has been introduced to enhance their detection responses in $LC-ESI-MS^n$ analysis. The aim of present study was to develop a sensitive method for identification and confirmation of nandrolone and its metabolite in horse urine incorporating pre-column derivatization using picolinic acid. The method consists of extraction of targeted steroid conjugates by solid phase extraction (SPE). The eluted steroid conjugates were hydrolysed by methanolysis and free steroids were recovered with liquid-liquid extraction. The resulting steroids were derivatized to form picolinoyl esters and identification was done using LC-ESI-MS/MS in positive ionization mode. The picolinated steroid adduct enhanced the detection levels in comparison to underivatized steroids.
Androgenic anabolic steroids (AASs) are synthetic derivatives of testosterone with a common structure containing cyclopentanoperhydrophenanthrene nucleus. Their use enhances the muscle building ...capacity and is beneficial during performance.
The AASs are one of the most abused group of substances in horse doping. Liquid chromatography tandem mass spectrometry (LC/MSn) has been successfully applied to the detection of anabolic steroids in biological samples. However, the saturated hydroxysteroids viz: nandrolone, 5α-estrane-3β, 17α-diol exhibit lower detection responses in electrospray ionisation (ESI) because of their poor ionisation efficiency. To overcome this limitation pre-column chemical derivatization has been introduced to enhance their detection responses in LC-ESI-MSn analysis. The aim of present study was to develop a sensitive method for identification and confirmation of nandrolone and its metabolite in horse urine incorporating pre-column derivatization using picolinic acid. The method consists of extraction of targeted steroid conjugates by solid phase extraction (SPE). The eluted steroid conjugates were hydrolysed by methanolysis and free steroids were recovered with liquid-liquid extraction. The resulting steroids were derivatized to form picolinoyl esters and identification was done using LC-ESI-MS/MS in positive ionization mode. The picolinated steroid adduct enhanced the detection levels in comparison to underivatized steroids. KCI Citation Count: 0
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.
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 35−36 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 < 0.05 and met ≥ 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.
AimTo investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. MethodsChimeric 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. ResultsCYP17A1 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. ConclusionIn 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.
•20-hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent manner.•NOS3 mRNA and proteins are upregulated by the compound in human endothelial cells•NOS3 mRNA is upregulated by the ...compound in humanized liver tissues•The mechanism does not appear to involve activation of estrogen ER-β receptors•This effect is not specific to muscle arterioles, which could limit its benefit
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.
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.
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.
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.
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.
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.
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Dried blood spots have recently been approved by the World Anti-Doping Agency as an alternative biological matrix for testing of doping substances. However, their use is limited to the detection of ...non-threshold compounds without a Minimum Reporting Level due to the numerous issues related to quantitative analyses and the limitation on testing capabilities of a haemolysed matrix.
In this study androstenedione, testosterone and IGF-1 were longitudinally monitored in four different blood matrices to evaluate the potential of liquid capillary blood as an alternative matrix for quantitative determination in doping control analysis.
The analytical protocols developed to pretreat 20 μL of the blood matrices selected were based: i) for testosterone and androstenedione, on supported liquid extraction for liquid blood matrices, and on ultrasonication in the presence of methanol for dried blood matrices; ii) for IGF-1, proteins precipitation followed by evaporation of the supernatant was used to pretreat both liquid and dried blood matrices. The detection for all the target analytes was performed using liquid chromatography coupled to mass spectrometry. The analytical workflows, once optimized, were fully validated according to the requirements of World Anti-Doping Agency and ISO 17025 standard and used for the analysis of venous (serum) and capillary (liquid plasma and dried whole blood collected using either volumetric or non-volumetric devices) blood samples collected from 7 healthy subjects.
The validation results showed satisfactory performance as related to specificity, sensitivity, matrix effects, linearity, accuracy, and precision in all the blood matrices evaluated despite the limited volume of sample used. The analysis of the different blood matrices collected from the subjects showed non-significant differences between the levels of testosterone and androstenedione measured in dried (fixed volume collected) and liquid matrices. An acceptable underestimation (lower than 15 %) was observed in capillary plasma compared to venous serum. The testosterone/androstenedione ratio was similar in all the blood matrices considered (bias lower than 5 %), indicating this parameter was not affected by either the blood matrix or collection device selected. For IGF-1, the levels measured in liquid blood matrices differed significantly (bias higher than 20 %) from those measured in dried whole blood matrices, suggesting haemolyzed blood might represent a challenge for the determination of macromolecules, mainly due to the complexity of the whole blood matrix in comparison to plasma/serum.
The outcomes of our study suggest that liquid capillary blood might open new avenues to blood microsampling in doping control field. It represents an efficient alternative to overcome the issues related to venous blood and dried blood spot sampling. Furthermore, it also allows greater frequency of blood sampling, with minor discomfort and without needing a phlebotomist, for analyses that can only be performed in blood samples, with an increased probability to detect and report Adverse Analytical Finding.
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•DBS was approved by the WADA as an alternative matrix only for qualitative determination.•The potential of liquid capillary blood as an alternative matrix for quantitative analysis was evaluated.•Selected pseudo-endogenous compounds were longitudinally monitored in four blood matrices.•Liquid capillary blood is an acceptable alternative to venous blood for quantitative determination.•Liquid capillary blood might open new avenues to blood microsampling in doping control field.
Abstract only Introduction: Fatty acids (FAs) have been of interest as biomarkers and as mechanistic signaling modulators of inflammation for the progression of coronary heart disease (CHD). Short ...chain FAs (SCFA) play a role in two inflammation mediating signaling mechanisms: inhibition of histone deactylase and activation of G-protein-coupled receptors. The omega-3 FAs are important in the generation of resolvins, which are active in the resolution of inflammation. Medium chain FAs (MCFA) play an important role in decreasing cholesterol by inhibiting bile acid reabsorption. In this study we investigated changes of FAs in CHD. Methods: Samples from the Qatar Cardiovascular Biorepository and Qatar Biobank (CHD n=1,001, controls n=2,999) were used for the quantitative analysis of serum FAs by liquid chromatography-mass spectrometry. Data were curated by Metabolon Inc. Standard quality control steps were performed. Logistic regression, adjusting for age, BMI, and sex, was conducted to test association between CHD and FAs. Results: Overall 289 lipid metabolites including 27 long chain FAs (LCFA), 9 MCFA, and one SCFA (caproate) were analyzed. The significant metabolites at P <0.05 were 25 LCFA (20 were higher in CHD), 7 MCFA (4 were higher in CHD), and one SCFA (higher in CHD, P =1.64х10 -87 ). MCFA heptanoate (7:0, P =1.67х10 -26 ), LCFA erucate (22:1n9, P =5.5х10 -8 ) and docosadienoate (20:2n6, P =1.25х10 -5 ) were higher in CHD than controls. Among the FAs that were lower in CHD, there were the long chain omega-3 FAs eicosapentaenoate (EPA, 20:5n3, P =1.6х10 -3 ), docosahexaenoate (DHA, 22:6n3, P =1.45х10 -10 ), and docosapentaenoate (DPA, 22:5n3, P =1.64х10 -11 ), and the MCFA 5-dodecenoate (12:1n7, P =3.5х10 -4 ) and 10-undecenoate (11:1n1, P =3.79х10 -11 ). Conclusions: Our results show that changes in the FAs profile of the CHD patients in this study may mechanistically contribute to the increased prevalence of inflammatory diseases such as CHD in the Middle East.
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