phytosterols and boldenone

Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated. We carried out a 5-week in vivo trial with both men (n=6) and women (n=6) and measured alpha-boldenone, beta-boldenone, boldione, androstenedione, beta-testosterone and alpha-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC-MS-MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL(-1) to 1.73 ng mL(-1), whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols. This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.

Topics: Adult; Anabolic Agents; Androstadienes; Androstenedione; Biotransformation; Epitestosterone; Female; Food Analysis; Gas Chromatography-Mass Spectrometry; Humans; Male; Middle Aged; Molecular Structure; Phytosterols; Tandem Mass Spectrometry; Testosterone; Young Adult

Collaborative microbial communities are ubiquitous in nature and exhibit appealing functions for enhanced production of natural products, which provides new possibility for biotechnology development. In this study, we bridged Mycobacterium neoaurum with Pichia pastoris to establish a step-wise biotransformation strategy for efficient biosynthesis of boldenone (BD) from phytosterol (PS). Firstly, the producing strains were rationally designed with overexpression of 3-ketosteroid-Δ1-dehydrogenase (KsdD) and 17β-hydroxysteroid dehydrogenase (17βHSD) in M. neoaurum and P. pastoris, respectively. Then, to shorten the total biotransformation process and provide reducing power, semi-batch fermentation strategy and glucose supplementation strategy were introduced at side-chain degradation stage and carbonyl reduction stage, respectively. Under the optimal transformation conditions, the productivity of BD was increased from 10% to 76% and the total biotransformation process was shortened by 41.7%, which is the shortest among the ever reported. Our results demonstrated an excellent biological strategy for production of many other valuable microbial products from bioresources.

Topics: 17-Hydroxysteroid Dehydrogenases; Biotransformation; Fermentation; Nontuberculous Mycobacteria; Oxidoreductases; Phytosterols; Pichia; Testosterone

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that β-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including β-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of β-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to β-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% β-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of β-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of β-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and β-Bol.

Topics: Amino Acids; Anabolic Agents; Androgens; Androstadienes; Androstenedione; Animals; Chromatography, High Pressure Liquid; Dietary Carbohydrates; Digestion; Fatty Acids, Volatile; Female; Horses; Male; Mycobacterium; Phytosterols; Steroids; Tandem Mass Spectrometry; Testosterone

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

Topics: Anabolic Agents; Androstenedione; Animal Feed; Animal Use Alternatives; Animals; Biosynthetic Pathways; Cattle; Chromatography, High Pressure Liquid; Crustacea; Diptera; Fungi; Larva; Microsomes; Phytosterols; Pleurotus; Substance Abuse Detection; Tandem Mass Spectrometry; Testosterone

Cholesterol is a well-known component in fats of animal origin and it also is the precursor of natural hormones. Phytosterols appear in plants and only differ slightly in structure from cholesterol. An important difference however is the low absorption in the gut of phytosterols and their saturated derivatives, the phytostanols. As a result, there is time for all kind of reactions in faecal material inside and outside of the gut. Determination of the abuse of natural hormones may be based on gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Abuse of natural hormones changes the 13C/12C ratio of some metabolites during a relatively long time. The formation of (natural) hormones in the gut may interfere with this method. Designer drugs are mainly known from sports doping. In animal fattening, designer drugs may be used as well. Small changes in the structure of (natural) hormones may lead to a new group of substances asking for new strategies for their detection and the constatation of their abuse.

Topics: Anabolic Agents; Animals; Cholesterol; Designer Drugs; Doping in Sports; Female; Gynecomastia; Hormones; Humans; Male; Mass Spectrometry; Phytosterols; Substance Abuse Detection; Testosterone; Veterinary Medicine

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves' faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer (Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and -stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10-0.14% (p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, beta-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and beta-boldenone at a significant (p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.

Topics: Anabolic Agents; Androstadienes; Animals; Artemia; Body Weight; Chemistry Techniques, Analytical; Chromatography, Liquid; Diptera; Larva; Mass Spectrometry; Models, Chemical; Phytosterols; Quality Control; Steroids; Testosterone

The residue profiles of 17alpha-/17beta-boldenone conjugated (17alpha/beta-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4 h after feeding and further from all the animals. Detectable amounts of 17alpha-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17alpha-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17beta-Bol and ADD were never detected in urine samples collected.

Topics: Anabolic Agents; Animal Feed; Animals; Calibration; Cattle; Chromatography, Liquid; Diet; Male; Mass Spectrometry; Milk; Models, Chemical; Phytosterols; Sterols; Testosterone; Time Factors

The presence of the anabolic steroid boldenone in animals has become a research topic as its occurrence is proposed to be a marker for illegal hormone administration. However, boldenone can also be formed from beta-sitosterol, a phytosterol present in animal feed, as well as from endogenous sources. The observations in animals together with the increased consumption of phytosterol-enriched foods in the Western population led the authors to the hypothesis that consumption of phytosterol-enriched foods might possibly lead to increased boldenone levels in humans. The authors performed a pilot study among female volunteers (n = 10) to investigate whether boldenone concentrations in urine were detectable after consumption of 25 g day(-1) of phytosterol-enriched margarines for 1 week. Urine samples were collected at days 0, 3 or 4, and 7. Urine of a sitosterolemia (a rare autosomal recessively inherited lipid metabolic disorder) patient was collected as a positive control case. No traces of boldenone were detected in either the volunteers or in the patient. In conclusion, there is no evidence of formation of boldenone in women after consumption of the recommended amount of phytosterol-enriched margarines.

Topics: Adult; Anabolic Agents; Chromatography, Liquid; Female; Humans; Margarine; Mass Spectrometry; Middle Aged; Phytosterols; Pilot Projects; Sterols; Substance Abuse Detection; Testosterone

Twenty-six veal calves were split into two groups and fed two milk replacers with a different content of phytosterols for 26 days; then, 14 calves (7 animals from each diet) were kept as controls and 12 calves (6 per diet) received daily, per os, a combination of 17beta-boldenone (17beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) for 38 days. The urinary elimination of 17 alpha-/17beta-boldenone conjugates (17 alpha/beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) was followed by liquid chromatography-tandem mass spectrometry from all of the animals until slaughtering. In urine from treated animals, 17 alpha-Bol concentrations, despite a great variability, were greater than 17beta-Bol, both detected always as conjugates. At days 1, 2, and 3, the mean urine concentration of 17 alpha-Bol was higher than 12 ng/mL. A remarkable decrease was observed during the following days, but the 17 alpha-Bol concentration was still higher than the attention level of 2 ng/mL in 58% of the samples; the concentration of 17beta-Bol was around the action level of 1 ng/mL; two days after treatment withdrawal, no 17beta-Bol was detected in the urine. In urine from control animals, the 17 alpha-Bol concentration was strictly related to the phytosterol content of the diet, while, in urine from treated animals, the much higher 17 alpha-Bol levels were not modified by the production from diet precursors. The results confirmed that a 17 alpha-Bol level higher than 2 ng/mL should be considered as evidence of suspected illegal treatment and that the urinary excretion of 17beta-Bol is due to exogenous administration of 17beta-Bol. The discontinuous rate of elimination of both 17 alpha- and 17beta-Bol, despite the daily administration of 17beta-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.

Topics: Anabolic Agents; Androstadienes; Animals; Cattle; Diet; Male; Milk Substitutes; Phytosterols; Testosterone