Doping in sport—2. Quantification of the impurity 19-norandrostenedione in pharmaceutical preparations of norethisterone

The finding of measurable amounts of 19-norandrostenedione in norethisterone tablets prompted us to develop an assay to quantify this steroid. 19-Norandrostenedione is an anabolic steroid whose use in sport is prohibited by the World Anti-Doping Agency (WADA). The assay was developed using isotope dilution and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 19-norandrostenedione in norethisterone formulations, with [3,4-¹³C₂]-19-norandrostenedione as the internal standard. The results showed amounts up to 1.01 ± 0.01 μg (mean ± S.E.M.) per tablet in those containing 5 mg of norethisterone or norethisterone acetate (0.02%, w/w) and up to 0.5 ± 0.01 μg (mean ± S.E.M.) per tablet (0.05%, w/w) in oral contraceptive tablets containing 0.35-1.5 mg of norethisterone or norethisterone acetate. No tablet tested exceeded the British Pharmacopoeia limit of 0.1% for this impurity.     

Doping in sport—1. Excretion of 19-norandrosterone by healthy women, including those using contraceptives containing norethisterone

19-Norandrosterone (19-NA) is the principal urinary metabolite of the anabolic steroid nandrolone and its prohormones. The administration of these 19-nor androgens is prohibited in sport by the World Anti-Doping Agency (WADA) but, even so, adverse findings for 19-NA continue to be commonly reported. Little is known about the urinary concentrations of 19-NA that can occur in women who are not using anabolic steroids, including those using oral contraceptives containing the 19-nor progestogen norethisterone. In 2004, WADA lowered the reporting threshold for 19-NA for females from 5 to 2 ng/mL. The lack of any substantial data on 19-NA excretion in women prompted this large-scale investigation. In this investigation, single untimed urines collected from 1202 female volunteers, 38 of whom were taking norethisterone containing contraceptives, were analysed for 19-NA. None of the women was a competitive athlete and pregnancy had been excluded by a urinary test for human chorionic gonadotropin (hCG). Only one sample exceeded the 19-NA reporting threshold having a concentration of 4.1 ng/mL. This sample was from a user of a norethisterone-containing contraceptive.     

Evidence for the presence of endogenous 19-norandrosterone in human urine

In 1997, in the scope of antidoping control in sport, a not inconsiderable number of urine analysed by official laboratories revealed the presence of 19-nortestosterone (19-NT: 17β-hydroxyestr-4-en-3-one) metabolites: 19-norandrosterone (19-NA: 3α-hydroxy-5α-estran-17-one) and 19-noretiocholanolone (19-NE: 3α-hydroxy-5β-estran-17-one). These repeated results on a short period of time generated some investigations and especially the verification of the possible production of these metabolites by an unknown endogenous route in adult entire male. Some experiences were led on different persons known to be non-treated with steroids and more precisely with nandrolone. Extractive methods were developed focusing on their selectivity, i.e. searching to eliminate at best matrix interferences from the target analytes. Gas chromatography coupled to mass spectrometry (quadrupole and magnetic instruments) was used to detect, identify and quantify the suspected signals. Two types of derivatization (TMS and TBDMS), a semi-preparative HPLC as well as co-chromatography proved unambiguously the presence, in more than 50% of the analysed urine (n=40), of 19-NA at concentrations between 0.05 and 0.60 ng/ml. 19-NE was not detected with the developed methods (LOD<0.02 ng/ml). Experiments led on athletes showed that after a prolonged intense effort, the 19-NA concentration can be increased by a factor varying between 2 and 4. Even if some complementary researches have to be done in order to determine the maximal physiological level of 19-NA and 19-NE, these results should considerably change the strategy of antidoping laboratories.     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.     

Determination of nandrolone and metabolites in urine samples from sedentary persons and sportsmen

Metabolites of nandrolone were determined in the urine of several sportsmen, sedentary and post-menopausal women by capillary gas chromatography–mass spectrometry quadrupole (GC–MS) and capillary gas chromatography mass–mass spectrometry ion trap (GC–MS–MS) methods. The method employed was GC–EI-MS with 17α-methyltestosterone as internal standard with ethyl ether extraction prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405 and 420 for the nandrolone metabolites, and 418 and 403 for nandrolone derivative. Recovery for nandrolone, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) was 97.20, 94.17 and 95.54%, respectively. Detection limits for nandrolone, 19-NA and 19-NE were 0.03, 0.01 and 0.06 ng/ml. Metabolites of nandrolone (19-NA and 19-NE) were found in 12.5% (n=40) of sportsmen and 40% (n=10) of post-menopausal women.     

Profiling of 19-norandrosterone sulfate and glucuronide in human urine: Implications in athlete's drug testing

19-Norandrosterone (19-NA) as its glucuronide derivative is the target metabolite in anti-doping testing to reveal an abuse of nandrolone or nandrolone prohormone. To provide further evidence of a doping with these steroids, the sulfoconjugate form of 19-norandrosterone in human urine might be monitored as well. In the present study, the profiling of sulfate and glucuronide derivatives of 19-norandrosterone together with 19-noretiocholanolone (19-NE) were assessed in the spot urines of 8 male subjects, collected after administration of 19-nor-4-androstenedione (100 mg). An LC/MS/MS assay was employed for the direct quantification of sulfoconjugates, whereas a standard GC/MS method was applied for the assessment of glucuroconjugates in urine specimens. Although the 19-NA glucuronide derivative was always the most prominent at the excretion peak, inter-individual variability of the excretion patterns was observed for both conjugate forms of 19-NA and 19-NE. The ratio between the glucuro- and sulfoconjugate derivatives of 19-NA and 19-NE could not discriminate the endogenous versus the exogenous origin of the parent compound. However, after ingestion of 100 mg 19-nor-4-androstenedione, it was observed in the urine specimens that the sulfate conjugates of 19-NA was detectable over a longer period of time with respect to the other metabolites. These findings indicate that more interest shall be given to this type of conjugation to deter a potential doping with norsteroids.     

A simplified procedure for GC/C/IRMS analysis of underivatized 19-norandrosterone in urine following HPLC purification

Nandrolone and/or its precursors are included in the World Anti-doping Agency (WADA) list of forbidden substances and methods and as such their use is banned in sport. 19-Norandrosterone (19-NA) the main metabolite of these compounds can also be produced endogenously. The need to establish the origin of 19-NA in human urine samples obliges the antidoping laboratories to use isotope ratio mass spectrometry (IRMS) coupled to gas chromatography (GC/C/IRMS). In this work a simple liquid chromatographic method without any additional derivatization step is proposed, allowing to drastically simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis. The purity of the extracts was verified by parallel analysis by gas chromatography coupled to mass spectrometry with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision of ±0.3‰ and between assay precision of ±0.4‰). The method has been tested with samples obtained after the administration of synthetic 19-norandrostenediol and samples collected during pregnancy where 19-NA is known to be produced endogenously. Twelve drugs and synthetic standards able to produce through metabolism 19-NA have shown to present δ¹³C values around −29‰ being quite homogeneous (−28.8 ± 1.5; mean ± standard deviation) while endogenously produced 19-NA has shown values comparable to other endogenous produced steroids in the range −21 to −24‰ as already reported. The efficacy of the method was tested on real samples from routine antidoping analyses.     

Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.     

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.     

Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and beta-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography-mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NA(g+s)>19-NE(g+s)>19-NEA(s). The concentration profiles of these three metabolites in urine peaked between 2 to 12h post-administration and declined thereafter until approximately 72-96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NA(s+g)+19-NE(s+g)+19-NEA(s)) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol and 19-nor-5-androstene-3beta,17beta-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.     

The synthesis of 19-norandrostenedione from dehydroepiandrosterone in equine placenta is inhibited by aromatase inhibitors 4-hydroxyandrostenedione and fadrozole

19-Norandrostenedione was synthesized in vitro from dehydroepiandrosterone by explants of equine full-term placenta. The synthesis of 19-norandrostenedione was inhibited by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole.     

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.     

Doping in sports and its spread to at-risk populations: an international review

Doping is now a global problem that follows international sporting events worldwide. International sports federations, led by the International Olympic Committee, have for the past half century attempted to stop the spread of this problem, with little effect. It was expected that, with educational programs, testing, and supportive medical treatment, this substance-abusing behavior would decrease. Unfortunately, this has not been the case. In fact, new, more powerful and undetectable doping techniques and substances are now abused by professional athletes, while sophisticated networks of distribution have developed. Professional athletes are often the role models of adolescent and young adult populations, who often mimic their behaviors, including the abuse of drugs. This review of doping within international sports is to inform the international psychiatric community and addiction treatment professionals of the historical basis of doping in sport and its spread to vulnerable athletic and non-athletic populations.     

COVID-19 impact on the diagnosis of Inborn Errors of Metabolism: Data from a reference center in Brazil

The COVID-19 pandemic led to the reorganization of health care in several countries, including Brazil. Inborn Errors of Metabolism (IEM) are a group of rare and difficult to diagnose genetic diseases caused by pathogenic variants in genes that code for enzymes, cofactors, or structural proteins affecting different metabolic pathways. The aim of this study was to evaluate how COVID-19 affected the diagnosis of patients with IEM during the first year of the pandemic in Brazil comparing two distinct periods: from March 1^st, 2019 to February 29^th, 2020 (TIME A) and from March 1^st, 2020 to February 28^th, 2021 (TIME B), by the analysis of the number of tests and diagnoses performed in a Reference Center in South of Brazil. In the comparison TIME A with TIME B, we observe a reduction in the total number of tests performed (46%) and in the number of diagnoses (34%). In both periods analyzed, mucopolysaccharidoses (all subtypes combined) was the most frequent LD suspected and/or confirmed. Our data indicates a large reduction in the number of tests requested for the investigation of IEM and consequently a large reduction in the number of diagnoses caused by the COVID-19 pandemic leading to a significant underdiagnosis of IEM.     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.     

Multiplexed LC‐MS/MS analysis of horse plasma proteins to study doping in sport

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC‐MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race‐horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis‐driven discovery of doping biomarker candidates.     

Ablation of Succinate Production from Glucose Metabolism in the Procyclic Trypanosomes Induces Metabolic Switches to the Glycerol 3-Phosphate/Dihydroxyacetone Phosphate Shuttle and to Proline Metabolism

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD⁺/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/^RNAiFAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.     

pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent K_m of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.