Non-targeted LC-MS based metabolomics analysis of the urinary steroidal profile

The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis

Influence of Indomethacin on Steroid Metabolism: Endocrine Disruption and Confounding Effects in Urinary Steroid Profiling of Anti-Doping Analyses

Anabolic androgenic steroids (AAS) are prohibited as doping substances in sports by the World Anti-Doping Agency. Concentrations and concentration ratios of endogenous AAS (steroid profile markers) in urine samples collected from athletes are used to detect their administration. Certain (non-prohibited) drugs have been shown to influence the steroid profile and thereby sophisticate anti-doping analysis. It was shown in vitro that the non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits selected steroid-biotransformations catalyzed by the aldo-keto reductase (AKR) 1C3, which plays a key role in the endogenous steroid metabolism. Kinetic parameters for the indomethacin-mediated inhibition of the AKR1C3 catalyzed reduction in etiocholanolone were determined in vitro using two comparing methods. As NSAIDs are very frequently used (not only) by athletes, the inhibitory impact of indomethacin intake on the steroid metabolism was evaluated, and steroid profile alterations were detected in vivo (one male and one female volunteer). Significant differences between samples collected before, during or after the intake of indomethacin for selected steroid profile markers were observed. The presented results are of relevance for the interpretation of results from doping control analysis. Additionally, the administration of NSAIDs should be carefully reconsidered due to their potential as endocrine disruptors

Editorial: OMICS-based approaches in sports research volume II.

OMICS approaches, including genomics, epigenomics, transcriptomics, proteomics, and metabolomics, continue to provide invaluable tools for better understanding of the molecular mechanisms underlying various physiological and pathological functions in health and disease. The rapid advancement of these tools and emergence of new ones is progressively filling the gap in our understanding of the complex networks that determine the structure, function, and dynamics of organisms. These advancements have greatly empowered discoveries of novel diagnostic and prognostic biomarkers as well as therapeutic targets, and provided a better guidance to precision and personalized medicine

Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a “dilute and inject” approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories

No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism

In‐depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3‐keto‐4‐ene hydroxy steroids

Rationale: The aromatase inhibitor formestane (4‐hydroxyandrost‐4‐ene‐3,17‐dione) is included in the World Anti‐Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2‐, 6‐ and 11‐hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI‐MS) for enhanced confidence in detection and selectivity for determination. Methods: Established derivatization protocols to introduce [2H9]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3‐keto‐4‐ene structure. Formestane was additionally labelled with H218O to obtain derivatives doubly labelled with [2H9]TMS and 18O. GC/EI‐MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high‐resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments. Results: Subclass‐specific fragment ions include m/z 319 for the 6‐hydroxy and m/z 219 for the 11‐hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2‐ and 4‐hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2‐, 4‐ and 6‐hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS‐derived methyl radical and a neutral loss of (CH3)2SiO. Further common fragments were also elucidated. Conclusions: With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18O‐labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2‐ and 4‐hydroxy compounds

Metabolic GWAS of elite athletes reveals novel genetically-influenced metabolites associated with athletic performance

Genetic research of elite athletic performance has been hindered by the complex phenotype and the relatively small effect size of the identified genetic variants. The aims of this study were to identify genetic predisposition to elite athletic performance by investigating genetically-influenced metabolites that discriminate elite athletes from non-elite athletes and to identify those associated with endurance sports. By conducting a genome wide association study with high-resolution metabolomics profiling in 490 elite athletes, common variant metabolic quantitative trait loci (mQTLs) were identified and compared with previously identified mQTLs in non-elite athletes. Among the identified mQTLs, those associated with endurance metabolites were determined. Two novel genetic loci in FOLH1 and VNN1 are reported in association with N-acetyl-aspartyl-glutamate and Linoleoyl ethanolamide, respectively. When focusing on endurance metabolites, one novel mQTL linking androstenediol (3alpha, 17alpha) monosulfate and SULT2A1 was identified. Potential interactions between the novel identified mQTLs and exercise are highlighted. This is the first report of common variant mQTLs linked to elite athletic performance and endurance sports with potential applications in biomarker discovery in elite athletic candidates, non-conventional anti-doping analytical approaches and therapeutic strategies

Routine application of SFC-MS in doping control: Analysis of 3 × 1000 urine samples using three different SFC-MS instruments

Supercritical fluid chromatography-mass spectrometry (SFC-MS) has proved to be a beneficial tool for sample analysis for a wide variety of compounds and, as such, has recently gained the attention of the anti-doping community. We have tested the applicability of SFC-MS for routine doping control analysing approximately 3 × 1000 identical anti-doping samples utilising SFC-MS instruments from three different vendors: Agilent Technologies, Waters Corporation and Shimadzu Corporation. A ‘dilute and inject’ approach either without or after hydrolysis of glucuronide metabolites was applied. Most of the compounds included in our study demonstrated excellent chromatography, whereas some showed co-elution with endogenous interferences requiring MS discrimination. Retention times typically were very stable within batches (%CV ≤ 0.5%), although this appeared to be analyte and column dependent. Chromatographic peak shape was good (symmetrical) and stable over the period of the testing without any change of column. Our results suggest that SFC-MS is a sensitive, reproducible and robust analytical tool ready to be used in anti-doping laboratories alongside the currently applied techniques such as gas and liquid chromatography coupled to mass spectrometry. Even if instruments are designed slightly differently, all three setups demonstrated their fitness for the purpose in anti-doping testing

High‐level performances following low altitude training and tapering in warm environments in elite racewalkers

Current guidelines for prolonged altitude exposure suggest altitude levels ranging from 2000 to 2500 m to optimize an increase in total hemoglobin mass (Hbmass). However, natural low altitude locations (&lt;2000 m) remain popular, highlighting the interest to investigate any possible benefit of low altitude camps for endurance athletes. Ten elite racewalkers (4 women and 6 men) underwent a 4-week "live high-train high" (LHTH) camp at an altitude of 1720 m (P &lt;sub&gt;I&lt;/sub&gt; O &lt;sub&gt;2&lt;/sub&gt; = 121 mmHg; 20.1°C; 67% relative humidity [RH]), followed by a 3-week tapering phase (20 m; P &lt;sub&gt;I&lt;/sub&gt; O &lt;sub&gt;2&lt;/sub&gt; = 150 mmHg; 28.3°C; 53% RH) in preparation for the World Athletics Championships (WC). Venous blood samples were withdrawn weekly during the entire observation period. In addition, blood volumes were determined weekly by carbon monoxide rebreathing during altitude exposure and 2 weeks after return to sea level. High-level performances were achieved at the WC (five placings among the Top 10 WC races and three all-time career personal bests). A slight but significant increase in absolute (+1.7%, p = 0.03) and relative Hbmass (+2.3%, p = 0.02) was observed after 4-week LHTH. In addition, as usually observed during LHTH protocols, weekly training distance (+28%, p = 0.02) and duration (+30%, p = 0.04) significantly increased during altitude compared to the pre-LHTH period. Therefore, although direct causation cannot be inferred, these results suggest that the combination of increased training load at low altitudes with a subsequent tapering period in a warm environment is a suitable competition-preparation strategy for elite endurance athletes

Quinoa as phytopharmaceutical? Urinary elimination of ecdysterone after consumption of quinoa alone and in combination with spinach

The phytosteroid ecdysterone is classified as an anabolic agent and has been included on the monitoring list of the World Anti-Doping Agency since 2020. Therefore, the consumption of food rich in ecdysterone, such as quinoa and spinach, is the focus of a lively debate. Thus, the urinary excretion of ecdysterone and its metabolites in humans was investigated following quinoa consumption alone and in combination with spinach. Eight participants (four male and four female) were included, and they ingested 368 ± 61 g cooked quinoa alone and in combination with 809 ± 115 g spinach after a washout. Post-administration urines were analyzed by LC-MS/MS. After intake of both preparations, ecdysterone and two metabolites were excreted in the urine. The maximum concentration of ecdysterone ranged from 0.44 to 5.5 µg/mL after quinoa and from 0.34 to 4.1 µg/mL after quinoa with spinach. The total urinary excreted amount as parent drug plus metabolites was 2.61 ± 1.1% following quinoa intake and 1.7 ± 0.9% in combination with spinach. Significant differences were found in the total urinary excreted amount of ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone. Only small portions of ecdysterone from quinoa and the combination with spinach were excreted in the urine, suggesting that both quinoa and spinach are poor sources of ecdysterone in terms of bioavailability