Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design

Abstract

In the European Union, the use of thyreostats for animal fattening purposes has been banned and monitoring plans have been established to detect potential abuse. However, this is not always straightforward as thyreostats such as thiouracil may also have a semi-endogenous origin. Therefore, this study aimed at defining urinary metabolites, which may aid in defining the origin of detected thiouracil. Hereto, a parallel-like randomized in vivo study was conducted in which calves (n = 8) and cows (n = 8) were subjected to either a control treatment, rapeseed-enriched diet to induce semi-endogenous formation, or thiouracil treatment. Urine samples (n = 330) were assessed through metabolic fingerprinting, employing liquid-chromatography and Q-ExactiveTM Orbitrap mass spectrometry. Urinary fingerprints comprised up to 40,000 features whereby multivariate discriminant analysis was able to point out significant metabolome differences between treatments (Q2(Y) ≥ 0.873). Using the validated models, a total of twelve metabolites (including thiouracil) were assigned marker potential. Combining these markers into age-dependent biomarker panels rendered a tool by which sample classification could be improved in comparison with thiouracil-based thresholds, and this during on-going thiouracil treatment (specificities ≥ 95.2% and sensitivities ≥ 85.7%), post-treatment (sensitivities ≥ 80% for ≥ 24 h after last administration), and simulated low-dose thiouracil treatment (exogenous thiouracil below 30 ng μL-1). Moreover, the metabolic relevance of revealed markers was supported by the suggested identities, for which a structural link with thiouracil could be determined in most cases. The proposed biomarker panels may contribute to a more justified decision-making in monitoring thiouracil abuse.

A role for metabolomics in the antidoping toolbox?

“…Glucuronidated metabolites of the original drug are reported in serum for ractopamine, 104 and for estradiol and nortestosterone in urine 110 . Similarly, the administration of thiouracil shows several derivative metabolites in bovine urine 103 while administration of trenbolone and estradiol results in detectable derivative metabolites in bovine serum 112 . All these markers are the result of the liver metabolic transformation and are expected.…”

In vivo Administration of Testosterone Propionate in Cattle Analyzed by High Performance Liquid Chromatography-Tandem Mass Spectrometry: An Enzymatic Hydrolysis Study and Drug Abuse Issues

“…To monitor these substances from a surveillance perspective, a range of matrices must be included in regulatory plans, and urine is generally the matrix of choice. [1][2][3] However, analytical results concerning to the presence of endogenously occurring steroids (boldenone, estradiol, nortestosterone, and testosterone) demand additional strategies such as the use of gas chromatography-combustion-isotope ratio mass spectrometer systems (GC-C-IRMS), 4 omic approaches, 5,6 intact steroid ester detection in hair/plasma, 7,8 or longitudinal monitoring of biomarkers. 9,10 As limiting factors, the first alternative has poor sensitivity and high acquisition/maintenance costs, which prevents its widespread use.…”

Discrimination between the exogenous and endogenous origin of thiouracil in farm animals, the final chapter?

“…Data for intentional abuse of thiouracil is scarce. Two different studies (Wauters et al 2017;Van Meulebroek et al 2018) were performed to determine threshold levels based on animal experiments whereby bovines were treated with thiouracil. One of these studies was also aiming to identify relevant biomarkers for thiouracil abuse (Van Meulebroek et al 2018).…”

“…Two different studies (Wauters et al 2017;Van Meulebroek et al 2018) were performed to determine threshold levels based on animal experiments whereby bovines were treated with thiouracil. One of these studies was also aiming to identify relevant biomarkers for thiouracil abuse (Van Meulebroek et al 2018). In the two studies conducted during which animals were treated with thiouracil, the concentration of thiouracil in urine was very high during administration (>10.000 µg/L).…”

“…After the treatment period, the concentration of the thiouracil dropped rapidly. Several biomarkers were also tentatively identified (Van Meulebroek et al 2018), and a decision model was built combining these biomarkers to determine whether an animal has been treated with thiouracil or not. There is no information available on exogenous levels after the administration of thiouracil to species other than bovine.…”