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

Thiouracil (2-thiouracil) is a thyreostatic compound that can be used as an illegal growth promoter. In bovine, porcine and other farm animals, low concentrations of thiouracil are detected in urine. There is much debate on which concentrations can be considered to originate from feed ('natural') and which concentrations are caused by the illegal administration of thiouracil for growth-promoting purposes. Currently, a threshold value of 10 µg/L in urine is applied. The threshold value is based on epidemiological data. Data on thiouracil from animals treated with thiouracil is scarce. We conducted a study whereby animals were fed with rapeseed, rapeseed with thiouracil, or regular feed with thiouracil (low and high concentration). It was determined that administration of thiouracil leads to concentrations higher than the current 10 µg/L threshold of thiouracil and its metabolites in urine during treatment. Animals fed with rapeseed showed higher thiouracil concentrations than the control group, mostly above 10 µg/L and in some cases above 30 µg/L. In the discovery study, several biomarkers for thiouracil treatment were tentatively identified and confirmed with reference standards. One metabolite was identified as indicative for thiouracil abuse, namely 6-methyl-thiouracil. Another metabolite, 4-thiouracil, was indicative for endogenous formation and did not increase during 2-thiouracil treatment. 6-Methyl-thiouracil was not found in urine samples from the Dutch routine control programmes that contained (endogenous) 2-thiouracil above the threshold value. However, 4-thiouracil was found at high concentrations in the same samples when 2-thiouracil was present. This study's overall conclusion is that the threshold value for thiouracil in bovine urine samples should be set at 10 µg/L and for porcine urine samples at 30 µg/L. Also, confirmation of 6-methyl-thiouracil and 4-thiouracil should be used as indicators for exogenous or endogenous origin in routine control monitoring programmes.

Analysis of thyreostats in bovine feces using ultra high performance liquid chromatography with tandem mass spectrometry.

A method based on ultra-high performance liquid chromatography was developed and validated to detect six thyreostatic compounds: tapazole, thiouracil, methylthiouracil, dimethylthiouracil, propylthiouracil, and phenylthiouracil in faeces of bovine. Thyreostats were extracted from the matrix with a mixture of methanol and buffer (pH = 8). Next step was derivatization of analytes with 3-iodobenzylbromide. The liquid chromatographic separation of derivatives was obtained on a SB-C18 column (50 × 2.1 mm; 1.8 µm, Agilent) with gradient elution using a mobile phase consisting of acetonitrile/0.1% acetic acid within 7.5 min. The analysis was performed on a Shimadzu NEXERA X2 ultra-high performance liquid chromatograph with triple quadrupole MS 8050 instrument operating in positive electrospray ionization mode. Depending on the target compound, two or three diagnostic signals (selected reaction monitoring transitions) were monitored. The procedure was validated according to the Commission Decision 2002/657/EC. Recovery and repeatability met the performance criteria specified by this document for banned compounds. The recovery ranged from 97.5 to 110.5%, and repeatability did not exceed 14.1%. Decision limits and detection capabilities were below 10 µg/kg. The highest decision limits and detection capabilities concentrations were observed for phenylthiouracil of 3.48 and 6.96 µg/kg, respectively.

The effect of diet enriched with rapeseed meal on endogenous thiouracil contents in urine and milk of cattle.

Thyreostatic compounds, such as thiouracil, are orally active drugs that can be used to increase the weight of cattle before slaughter. Due to potentially teratogenic and carcinogenic effects of their residues on public health, the use of thyreostats in animal production has been banned in the European Union since 1981. Systematic detection of low concentrations of thiouracil in the urine of livestock in many countries is believed to be of endogenous origin due to the use of Brassicaceae plants in the animal diet. Therefore, the purpose of the study was to determine the effects of diets rich in rapeseed meal on formation of thiouracil in urine and milk of dairy cows. For two weeks three cows were subjected to a diet supplemented with rapeseed at 30%, compared to the control cattle diet which contained up to 11% rapeseed. During the experiment, samples of urine and milk were collected and analysed by LC-MS/MS. The increase and decrease of thiouracil concentration in urine samples in different animals was individual and cyclic. The highest concentration of natural thiouracil determined in urine was 3.61 µg l-1. It has been found that endogenous thiouracil exists in two tautomeric forms. A few days of storage of frozen urine samples affected the stability of natural thiouracil, whereas an acidic medium improved the stability of the compound and its isomer, which remained stable even after two months of storage at temperatures below -18°C. Due to the instability of thiouracil, urine samples upon sampling should be delivered to the laboratory as soon as possible or properly preserved. In milk samples, thiouracil was not found above the decision limit of the applied method of 0.63 µg l-1. Preliminary studies have shown that faecal examination for banned thiouracil can be a complementary test for urine samples, and may be helpful in determining the origin of the compound present in urine.

Discrimination between Synthetically Administered and Endogenous Thiouracil Based on Monitoring of Urine, Muscle, and Thyroid Tissue: An in Vivo Study in Young and Adult Bovines.

Thiouracil (TU), synthesized for its thyroid-regulating capacities and alternatively misused in livestock for its weight-gaining effects, is acknowledged to have an endogenous origin. Discrimination between low-level abuse and endogenous occurrence is challenging and unexplored in an experimental setting. Therefore, cows (n = 16) and calves (n = 18) were subjected to a rapeseed-supplemented diet or treated with synthetic TU. Significant higher urinary TU levels were recorded after TU administration (

Energetic stabilities of thiolated pyrimidines on gold nanoparticles investigated by Raman spectroscopy and density functional theory calculations.

The adsorption structures of 2-thiocytosine (2TC) on gold surfaces were examined by means of vibrational Raman spectroscopy and quantum mechanical density functional theory calculations. The 1H-thione-amino form was calculated to be most stable among the six examined tautomers. The three plausible binding geometries of sulfur, pyrimidine nitrogen, and amino group binding modes were calculated to estimate the binding energies of the 1H-thione-amino form with six gold cluster atoms. Thiouracils including 2-thiouracil (2TU), 4-thiouracil (4TU), and 6-methyl-2-thiouracil (6M2TU) were also studied to compare their relative binding energies on gold atoms. The intracellular localization of a DNA base analog of 2TC on gold nanoparticles (AuNPs) in HeLa cells was identified by means of surface-enhanced Raman scattering. AuNPs were modified with 2TC by self-assembly. Our dark-field microscopy and z-depth-dependent confocal Raman spectroscopy indicated that 2TC-assembled AuNPs could be found inside cancer cells. On the other hand, we did not observe noticeably strong Raman peaks in the cases of thiouracils including 2TU, 4TU, and 6M2TU. This may be due to the additional amino group of 2TC, which can lead to a stronger binding of adsorbates on AuNPs.

Validation of a quantitative method using liquid chromatography coupled to multiple mass spectrometry for thiouracil in feedstuffs used in animal husbandry.

The use of thyreostatic drugs, like thiouracil (TU), in animal production has been banned for over three decades by the European Union, due to potential teratogenic and carcinogenic effects of its residues upon human consumption. Besides, thyreostats induce water retention in livestock, causing fallacious weight gain and deterioration of meat quality. Development of more competent analytical methods gave rise to sporadic TU detection in urine of untreated animals, questioning the actual synthetic origin TU. Research showed that TU can be formed upon digestion of Brassicaceae feeds in vivo and in vitro, which called for a means of differentiation between endogenous formation of TU and illicit administration. Therefore, in the present study, a routinely applicable liquid chromatography (LC) ion trap multiple mass spectrometry (MS(2)) method for TU analysis in animal feeds was optimised and validated, according to CD 2002/657/EC. A fractional factorial Plackett-Burman design was used to optimise the extraction procedure for TU from Brassicaceae and non-Brassicaceae feeds. This resulted in the discrimination of five influential factors (amount of feed, myrosinase, pH 7 buffer, 3-iodobenzyl bromide and elution solvent), for which the most optimal conditions were perfected. The limit of quantification for TU amounted 0.5 ng g(-1). The individual recoveries for TU ranged between 90.9 and 99.7%. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. ≤6.0 and ≤5.2%, respectively, for TU. Excellent linearity was proven based on determination coefficient (R(2) ≥ 0.99) and lack-of-fit test (F test, α = 0.05). Subsequently, a selection of feeds sampled during European national monitoring campaigns were evaluated with the present method showing concentrations ranging from 0.32 to 20.60 ng g(-1), demonstrating the relevance of the method in the analysis of TU from animal feeds.

One-step synthesis of large-scale graphene film doped with gold nanoparticles at liquid-air interface for electrochemistry and Raman detection applications.

We demonstrated a facile one-step synthesis strategy for the preparation of a large-scale reduced graphene oxide multilayered film doped with gold nanoparticles (RGO/AuNP film) and applied this film as functional nanomaterials for electrochemistry and Raman detection applications. The related applications of the fabricated RGO/AuNP film in electrochemical nonenzymatic H2O2 biosensor, electrochemical oxygen reduction reaction (ORR), and surface-enhanced Raman scattering (SERS) detection were investigated. Electrochemical data indicate that the H2O2 biosensor fabricated by RGO/AuNP film shows a wide linear range, low limitation of detection, high selectivity, and long-term stability. In addition, it was proved that the created RGO/AuNP film also exhibits excellent ORR electrochemical catalysis performance. The created RGO/AuNP film, when serving as SERS biodetection platform, presents outstanding performances in detecting 4-aminothiophenol with an enhancement factor of approximately 5.6 × 10(5) as well as 2-thiouracil sensing with a low concentration to 1 µM. It is expected that this facile strategy for fabricating large-scale graphene film doped with metallic nanoparticles will spark inspirations in preparing functional nanomaterials and further extend their applications in drug delivery, wastewater purification, and bioenergy.

Analysis of antithyroid drug residues in food animals.

In the late 1970s, concerns were raised regarding why antithyroid drugs were being administered to food animals to promote growth despite the fact that they had been implicated as being carcinogenic and teratogenic; the growth promotion process produced an inferior quality meat with increased water retention in the animal's gastrointestinal tract. An increased incidence of aplasia cutis (a characteristic scalp defect) in consumers in Spain was linked to an increased consumption of antithyroid-contaminated meat. Therefore, to protect human health, the EU banned the use of antithyroid drugs in food animal production in 1981. This article reviews the impact of this regulatory decision on the regulatory analysis of these compounds in foods of animal origin. It discusses the physiology of the thyroid gland, the chemistry of antithyroid drugs and critically evaluates the suitability of the analytical methods that have been developed and validated to support enforcement of the regulation.

Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay.

RNA levels are determined by the rates of both transcription and decay, and a mechanistic understanding of the complex networks regulating gene expression requires methods that allow dynamic measurements of transcription and decay in living cells with minimal perturbation. Here, we describe a metabolic pulse-chase labeling protocol using 4-thiouracil combined with large-scale RNA sequencing to determine decay rates of all mRNAs in Saccharomyces cerevisiae. Profiling in various growth and stress conditions reveals that mRNA turnover is highly regulated both for specific groups of transcripts and at the system-wide level. For example, acute glucose starvation induces global mRNA stabilization but increases the degradation of all 132 detected ribosomal protein mRNAs. This effect is transient and can be mimicked by inhibiting the target-of-rapamycin kinase. Half-lives of mRNAs critical for galactose (GAL) metabolism are also highly sensitive to changes in carbon source. The fast reduction of GAL transcripts in glucose requires their dramatically enhanced turnover, highlighting the importance of mRNA decay in the control of gene expression. The approach described here provides a general platform for the global analysis of mRNA turnover and transcription and can be applied to dissect gene expression programs in a wide range of organisms and conditions.

Feed or food responsible for the presence of low-level thiouracil in urine of livestock and humans?

In recent years, questions have been raised on the possible semi-endogenous status of the alleged xenobiotic thyreostatic drug thiouracil; thiouracil has been detected in the urine of various animals (livestock and domesticated) at concentrations between 1 and 10 µg L(-1) and also in human urine. Although several studies suggest Brassicaceae-derived feed as potential origin, no traces of thiouracil have been detected in feed so far. Therefore, the aim of this study was to elucidate the origin of thiouracil in the urine of livestock and humans. To this purpose various Brassicaceae feed and food sources (e.g., rapeseed, rapeseed coarse meal, cabbage, cauliflower, broccoli) were investigated for the presence of thiouracil. In addition, the impact of the Brassicaceae-related ß-thioglucosidase enzyme was evaluated. This myrosinase enzyme appeared to be crucial, because without its catalyzed hydrolysis no thiouracil could be detected in the various Brassicaceae-derived samples. Therefore, a sample pretreatment with incorporated enzymatic hydrolysis was developed after ensuring the quality performance of the extracted myrosinase mixture with a single-point glucose assay. Upon enzymatic hydrolysis and LC-MS(2) analysis, thiouracil was successfully detected in samples of traditional rapeseed, rapeseed-'00' variety coarse meal (values of erucic acid <2% and glucosinolates <25 µmol g(-1)), and rapeseed cake at 1.5, 1.6, and 0.4 µg kg(-1), respectively. As for the food samples, broccoli and cauliflower displayed thiouracil concentrations of 6.0 and <1.0 µg kg(-1), respectively. To the best of the authors' knowledge this study is the first to report the presence of naturally occurring thiouracil in feed and food samples. Future research should investigate the pathway of thiouracil formation and identify its possible precursors.

Simultaneous spectrophotometric determination of 2-thiouracil and 2-mercaptobenzimidazole in animal tissue using multivariate calibration methods: concerns and rapid methods for detection.

Two multivariate calibration methods, partial least squares (PLS) and principal component regression (PCR), were applied to the spectrophotometric simultaneous determination of 2-mercaptobenzimidazole (MB) and 2-thiouracil (TU). A genetic algorithm (GA) using partial least squares was successfully utilized as a variable selection method. The concentration model was based on the absorption spectra in the range of 200 to 350 nm for 25 different mixtures of MB and TU. The calibration curve was linear across the concentration range of 1 to 10 microg mL(-1) and 1.5 to 15 microg mL(-1) for MB and TU, respectively. The values of the root mean squares error of prediction (RMSEP) were 0.3984, 0.1066, and 0.0713 for MB and 0.2010, 0.1667, and 0.1115 for TU, which were obtained using PCR, PLS, and GA-PLS, respectively. Finally, the practical applicability of the GA-PLS method was effectively evaluated by the concurrent detection of both analytes in animal tissues. It should also be mentioned that the proposed method is a simple and rapid way that requires no preliminary separation steps and can be used easily for the analysis of these compounds, especially in quality control laboratories.

Determination of thyreostatics in animal feeds by CE with electrochemical detector.

A simple, rapid, reproducible and sensitive method based on CE with electrochemical detector was developed for the simultaneous determination of five thyreostatics including 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU) and methimazole (TAP) in animal feeds. A home-made wall-jet electrochemical detector with a 300 microm diameter platinum-disk-working electrode was equipped at the end of separation capillary and used to detect oxidation currents of these thyreostatics. Under the optimum experimental conditions, TU, MTU, PTU, PhTU and TAP could be well separated within 15 min at the separation voltage of 16 kV in 20 mmol/L sodium borate buffer (pH 9.2). The detection limits (S/N=3) of the five thyreostatics in animal feeds were found to be 7.6 microg/kg for TAP, 25 microg/kg for PTU, 15 microg/kg for PhTU, 18 microg/kg for TU and 20 microg/kg for MTU by the developed CE with electrochemical detector method coupled with solid-phase extraction sample pretreatment technique.

Analysis of thyreostatic drugs in thyroid samples by liquid chromatography tandem mass spectrometry: comparison of two sample treatment strategies.

A method based on ultra-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UPLC-MS/MS) for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with Decision 2002/657/EC. Sample extraction was evaluated in methanol and in ethyl acetate, the latter which gave better results. Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation chromatography (GPC). Recoveries ranged from 40% to 79% for the SPE approach and from 80% to 109% for GPC. Quantification was performed with blank tissue samples spiked with the analytes in the range 50-500microgkg(-1). 5,6-Dimethyl-2-thiouracil and 2-mercaptobenzimidazole-d(4) were used as internal standards. Decision limit (CCalpha) and detection capability (CCbeta) ranged from 1 to 15microgkg(-1) and from 6 to 25microgkg(-1), respectively. The accuracy of the method was calculated as percent error, which was less than 10%. The relative standard deviation in reproducibility conditions ranged between 2% and 14%.

A novel approach for simultaneous determination of 2-mercaptobenzimidazole and derivatives of 2-thiouracil in animal tissue by gas chromatography/mass spectrometry.

A novel approach for determination of 2-mercaptobenzimidazole (MBI) and other thyreostatic residues in animal tissues by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode was developed. The analytes in animal tissues (including hypothyroid, pork muscle and beef samples) were extracted by acetonitrile, and then purified by a matrix solid-phase dispersion (MSPD) procedure after the extraction residues had been dissolved in water. The thyreostatic residues were derivatized by pentafluorobenzyl bromide (PFBBr) under strong basic conditions and then N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) before GC/MS analysis. Different kinds of solid supports with various polarities for the MSPD procedure were investigated, and it was found that silica gel was suitable for the purpose. The average recoveries of the thyreostatic drugs in animal tissues ranged from 71.5-96.9% with the relative standard deviations below 10%. By using the developed method, the limits of detection were 10 microg/kg for MBI; 5 microg/kg for 6-phenyl-2-thiouracil; and 2 microg/kg for 2-thiouracil, 6-methyl-2-thiouracil and 6-propyl-2-thiouracil. The stability of the thyreostatic drugs in spiked animal tissues was tested, and the results showed that the thyreostatic drugs did not decompose within 3 months if the sample was stored in darkness below -20 degrees C.

Unambiguous identification of thiouracil residue in urine collected in non-treated bovine by tandem and high-resolution mass spectrometry.

Thyrostats are banned compounds in Europe since 1981 (directive 81/602/EC) because of their carcinogenic and teratogenic properties. However, the occurrence of thiouracil (TU) in bovine urines from national monitoring plans with quantifications in the range 1-10 microg . L(-1) occasionally raises the question of its origin which might either be the consequence of an illegal administration or the result of 'endogenous' production. In order to definitively and unambiguously identify the so-called thiouracil signal in non-treated bovine urines, independent mass spectrometry (MS) approaches have been used. Different reagents (3-IBBr, 3-BrBBr and PFBBr) were used to derivatise and to extract TU from urine samples and characterisation of the residues was performed by means of different MS approaches [LC/(ESI-)MS/MS, GC/(EI+)MS/MS and HRMS (EI and NCI)]. These combined strategies allowed for an independent and confident identification of TU in bovine urine samples collected from animals never treated with any thyrostatic drugs. This result is of prime importance for laboratories and risk managers involved in the field of forbidden growth promoters control: detection of TU residue in bovine urine will have to be carefully considered as a non-systematic proof of illegal administration.

An analytical method to screen for six thyreostatic drug residues in the thyroid gland and muscle tissues of food producing animals by liquid chromatography with ultraviolet absorption detection and liquid chromatography/mass spectrometry.

A method was developed and validated to screen for residues of the thyreostatic drugs, tapazole (TAP), mercaptobenzimidazole (MBI), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PrTU), and phenylthiouracil (PhTU) in bovine, equine, ovine, and porcine thyroid and muscle tissues at concentrations > or = 5 ng/g using 2-methoxy-mercaptobenzimidazole (MeMBI) and dimethylthiouracil (DMTU) as internal standards. In this method, the drugs were solvent extracted from thyroid and muscle tissue and cleaned up on an amino-propyl solid-phase extraction (SPE) cartridge. The unretained fraction containing TAP and MBI and the internal standard, MeMBI, was collected as Fraction 1. The retained fraction containing TU, MTU, PrTU, PhTU, and the internal standard, DMTU, was eluted with 3% acetic acid-isopropanol as Fraction 2. Fraction 1 was further cleaned up on an alumina B SPE cartridge and analyzed by gradient elution on a C18 high-performance liquid chromatography (HPLC) column with ultraviolet detection at wavelengths of 255 and 300 nm. Fraction 2 was taken to dryness, derivatized with 4-chloro-7-nitrobenzo-2-furazan at pH 8, and analyzed by gradient elution on a C18 LC column with mass spectrometry (MS) detection. Any "presumptive positive" test results were submitted for further analysis by LC/MS/MS. The validated method was applied to the analysis of over 300 thyroid tissue samples.

Flow injection analysis system equipped with a newly designed electrochemiluminescent detector and its application for detection of 2-thiouracil.

A new flow injection analysis (FIA) system equipped with an electrochemiluminescent (ECL) detector has been developed and applied for the ECL detection of 2-thiouracil. The FIA-ECL system used a specially designed flow-through ECL thin-layer cell to reduce the dead volume, the IR drop across the cell, and the probability of accumulation of gas bubbles in the cell. It was thus envisioned to improve the detection limit of the FIA-ECL method. After being established, the new FIA-ECL system was used to investigate the ECL response of 2-thiouracil in the presence of the ECL of Ru(bpy)3(2+). It was found that 2-thiouracil could enhance the ECL of Ru(bpy)3(2+) over a wide pH range (pH 4.0-12.0). A highly sensitive method for detection of 2-thiouracil in biological samples was developed by the new FIA-ECL system after optimizing several experimental conditions, such as the applied potential of the working electrode, the pH value of the aqueous solution, the flow rate of carrier solution, and the concentration of Ru(bpy)3(2+).

An ultrasensitive method for the determination of thiouracil and phenylthiouracil using capillary zone electrophoresis and laser-induced fluorescence detection.

This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of thiouracil (TU) and phenylthiouracil (PhTU) with high sensitivity (nanomolar range, i.e., attomoles detected). After derivatization with 5-iodoacetamidofluorescein, the analytes were separated by capillary zone electrophoresis using 20 mM phosphate buffer (pH 10.0) and quantified by fluorescence detection. The linearity range, precision, recovery, and detection limits were determined, and the method was shown to be applicable for the determination of TU and PhTU in spiked feed samples and urine.

Application of improved iodine-azide procedure for the detection of thiouracils in blood serum and urine with planar chromatography.

The application of iodine-azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapour for 5 s. The detection limits were established at pmol level. The factors depending on the detection limits were described. A comparison of iodine-azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated.