Metabolomics Approach to Anabolic Steroid Urine Profiling of Bovines Treated with Prohormones

Abstract

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

Metabolomics in food analysis: application to the control of forbidden substances

“…As no definitive and unique standard operation procedures currently exist, the comparative generation and analysis of data obtained from identical samples but acquired through different analytical conditions should help the interpretation of the information generated. Such developments relating to analysis and comparisons have recently been conducted in several laboratories, [12][13][14][15][16][17][18] leading to the formulation of in-house strategies for each of the steps of interest.…”

“…By combining the advantages of these new analytical tools, highthroughput strategies can be implemented to assess the metabolic profile of biological fluids with high sensitivity and selectivity (Figure 2b). Several applications have been reported in the context of anabolic practices detection in cattle based on ToF MS-finger printings [13,16,18] or Orbitrap. [14,18] Data processing and analysis…”

“…A number of metabolomics studies proving the relevance of the approach to discriminate situations involving anabolic-treated and non-treated animals have already been reported using urine as the matrix of choice. [13][14][15] Sample preparation generally involves a filtration followed by a normalization step to correct inter-and intra-individual variations of urine's concentration and allow generation of comparable fingerprints. Significant advances have been obtained in that context and the proof of concept is now widely accepted by the scientific community.…”

“…[12] In particular, metabolomics has recently drawn the attention of the scientific community in the detection of anabolic practices and several studies have indeed demonstrated the efficiency of such mass-spectrometric-based fingerprinting to discriminate animals that have been subjected to anabolic treatment from control animals. Several approaches have shown potential to discriminate through analysis of urine samples of animals treated with a range of anabolic formulations: DHEA and pregnenolone in bovines, [13] clenbuterol in calves, [14] recombinant equine growth hormone in horses, [15][16][17] and nandrolone and estradiol in calves. [18] Metabolomics is an emerging field of 'omics' research that focuses, in an untargeted mode, on large-scale and high-throughput measurement of small molecules (so-called metabolites) in biological matrices.…”

Natural Hormones in Food-Producing Animals:Legal Measurements and Analytical Implications

“…However, Rijk et al developed a metabolomic strategy of screening in urine for bovine animals treated with DHEA and pregnenolone. This methodology is an useful tool to track the abuse of these prohormones, but their reference levels remain unknown, making difficult their use in targeted analysis, at least until the metabolites of this non-focused approach are elucidated (Rijk et al, 2009). On the other hand, Dervilly-Pinel et al recently published a metabolomic study in urine for cattle treated with a combination of estradiol benzoate and nandrolone laureate.…”