Illegal Growth Promoters Research Articles

Pojavnost tiouracila u svinjećim i goveđim urinima prikupljenih na hrvatskim farmama

Thyrostats are a group of forbidden substances in food producing animals that increase water absorption in muscle tissue and the gastrointestinal tract of animals. These substances can be abused as illegal growth promoters as oral drugs on farms before slaughtering. The consequences of their illegal use on farm animals include higher yield though inferior meat quality, while these substances also pose a potential risk to human health, and so their application is banned in European Union Member States. Thiouracil (2-thiouracil) (TU) is the representative, and belongs to the group of thyrostats but also can be naturally present in the urine of farm animals fed with feed containing Brassicaceae species. The aim of this study was to monitor TU concentrations in pig and bovine urine in the period from 2015 to 2023. For this purpose, 391 urine samples were collected as a part of the National Residue Control Plan (NRCP) and TU concentrations were determined by liquid chromatography tandem mass spectrometry. TU was detected in 89 of 391 samples, or 22.76%. TU concentration ranged from 1.66 to 28.30 μg/L, and a statistically significant difference (P < 0.05) was determined in urine TU concentrations between pigs and bovines. Mean concentrations of TU varied by year, and ranged from 1.66 to 8.06 μg/L in pig urine and from 5.92 to 13.68 μg/L in bovine urine. None of the analysed urine samples contained TU concentration in excess of 30 μg/L, which is the cut-off value to distinguish potentially natural origin resulting from a cruciferous diet (Brassicaceae species). The results exclude the possibility of abuse of this substances in the livestock industry in the Republic of Croatia.

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

ABSTRACT 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.

Broad-spectrum detection of zeranol and its analogues by a colloidal gold-based lateral flow immunochromatographic assay in milk

Based on colloidal gold and broad-spectrum monoclonal antibody that binds to zeranol and its five analogues with high sensitivity, a lateral flow immunochromatographic assay (LFIA) in a competitive format was developed to specifically determine residues of zeranol, an illegal growth promoter in livestock. In this study, the assay had high sensitivity and was broad-spectrum only for zeranol and its five analogues, and the results were obtained within 10 min without needing sophisticated procedures. The cutoff values for zeranol and its five analogues were 10 ng/mL, and the IC50 values for zeranol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol and zearalenone were 1.250, 1.800, 1.775, 1.225, 1.709 and 1.319 ng/mL, respectively. The recovery rates were ranged from 85.6 to 93.9%, with the coefficient of variations less than 12.4%. The results demonstrated that the LFIA could be used for rapid, simultaneous, semi-quantitative and quantitative detection of residues of zeranol and its five analogous in milk.

Development of Highly Sensitive Immunosensor for Clenbuterol Detection by Using Poly(3,4-ethylenedioxythiophene)/Graphene Oxide Modified Screen-Printed Carbon Electrode.

Clenbuterol (CLB) is an antibiotic and illegal growth promoter drug that has a long half-life and easily remains as residue and contaminates the animal-based food product that leads to various health problems. In this work, electrochemical immunosensor based on poly(3,4-ethylenedioxythiophene)/graphene oxide (PEDOT/GO) modified screen-printed carbon electrode (SPCE) for CLB detection was developed for antibiotic monitoring in a food product. The modification of SPCE with PEDOT/GO as a sensor platform was performed through electropolymerization, while the electrochemical assay was accomplished while using direct competitive format in which the free CLB and clenbuterol-horseradish peroxidase (CLB-HRP) in the solution will compete to form binding with the polyclonal anti-clenbuterol antibody (Ab) immobilized onto the modified electrode surface. A linear standard CLB calibration curve with R2 = 0.9619 and low limit of detection (0.196 ng mL−1) was reported. Analysis of milk samples indicated that this immunosensor was able to detect CLB in real samples and the results that were obtained were comparable with enzyme-linked immunosorbent assays (ELISA).

Confirmation of β-agonist residues in bovine retina and liver using HPLC-MS/MS and evaluation of matrix-dependent problems

ABSTRACTβ-Agonists or β-adrenergic agonists are synthetic drugs sometimes used for therapeutic purposes in veterinary medicine. However, these compounds also belong to a class of illegal growth promoters that decrease the fat content in farm animals. The purpose of this study was to develop an analytical LC-MS/MS confirmatory method for the detection of nine β-agonists in bovine retina and liver tissues. The obtained CCα values ranged from 0.29 to 1.54 µg kg–1 in retina and from 0.03 to 0.18 µg kg–1 in liver, and CCβ values from 0.50 to 2.63 µg kg–1 and from 0.05 to 0.30 µg kg–1, respectively. Additionally, the importance of a matrix effect in these two tissues for the achievement of accurate detection of non-compliant samples was studied and discussed. It was concluded that the main characteristic of this phenomena is its considerable variability from one animal to another. Also, the total amount of matrix in the final extract can provide differences in the matrix-matched calibration line.

Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves

Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day–1 per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2–4 mg kg–1 i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1–T6; treatment and control group) and at four time points (T1–T4; comparison group) and stored at –80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics® software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment.

Development of an innovative microcantilever-based biosensor for 17β-estradiol detection in bovine muscles: preliminary results

17&beta;-estradiol is the most powerful substance with estrogenic effect, commonly used as illegal growth promoter in livestock production. To avoid health risks for consumers, sensitive, reliable and low-cost methods for quantification of extremely low concentrations of such carcinogenic residues in food are needed. Antibody-immobilised microcantilever resonators were proposed as innovative biosensors able to quantify an adsorbed target mass thanks to a shift in resonance frequency. Furthermore, the quantification of masses on the order of few picograms has recently shown to be successfully achievable with very high precision. In this study, we analysed the performance of our microcantilever sensors using extracted samples of bovine muscle from experimental animals, containing variable concentrations of 17&beta;-estradiol (HPLC-MS/MS tested). Preliminary data showed that treated animals are correctly revealed, exhibiting large negative frequency shifts. More experiments, though, are needed to obtain a correct quantification of 17&beta;-estradiol concentration.

The physiological way: Monitoring RNA expression changes as new approach to combat illegal growth promoter application

The use of growth-promoting agents in food-producing animals is forbidden in the European Union (EU). Therefore a strict control programme has been developed, detecting residues of all known growth-promoting agents using chromatographical methods in combination with mass spectrometry or immunoassays. New designed xenobiotic substances or hormone cocktails are difficult to identify with these methods and therefore the development of new sensitive test methods is important. A promising indirect approach is the detection of physiological effects of the administered growth promoters on the molecular level using 'omic' technologies. The analysis of the transcriptome on mRNA and miRNA level and thereby identifying biomarkers for the use of anabolic agents is one possible strategy for developing a new screening method. This paper describes the technologies available for gene expression profiling and summarizes the efforts made in the analysis of the transcriptome in order to identify potential gene expression biomarkers for the use of growth promoters in cattle.

Novel strategies for tracing the exposure of meat cattle to illegal growth-promoters

Official monitoring of residues in cattle throughout the European Union in 2007 found <0.2% non-compliance for the use of illegal growth-promoters (GPs), including sex steroids, corticosteroids and β-agonists. There is evidence, however, that these figures may underestimate the real incidence of GP abuse in meat cattle breeding. One source of evidence arises from the introduction of new detection strategies in response to the demand for safe and wholesome food. These strategies are based on the biological effects of the different GP classes in target species, with a focus on identifying reliable and cost effective biomarkers to improve detection methods. This review summarises the published data relating to experimental and field studies performed in meat cattle, emphasising the impact of the 'omic' technologies and bioinformatics to discover suitable biomarkers for residue surveillance. Further research is required before any potential biomarkers can be utilised for large scale high throughput screening tests.

Evaluation of indirect biomarkers for detecting corticosteroids used as illegal growth promoters in beef cattle

The histological status of the thymus, blood cortisol concentration and circulating neutrophil:lymphocyte ratio were evaluated in 349 slaughtered beef cattle, to assess the potential of these parameters as indirect biomarkers...

Multi-analyte approach for the determination of ng L−1 levels of steroid hormones in unidentified aqueous samples

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, β-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces.The aim of this study was to develop an analytical method for the determination of ngL−1 levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the ‘black’ market. For this, extraction was performed with Bakerbond C18 speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC–EI-MS2) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC–NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC–ESI-MS2). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ngL−1 level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

Characterization of the composition of bovine urine and its effect on the electrochemical analysis of the model mediator, p-aminophenol

The reliable identification of compounds such as illegal growth promoters in cattle is generally based on expensive gas chromatography–mass spectrophotometric analysis in urine, a method that does not allow on a large-scale screening. The use of simple, semi-quantitative electrochemical biosensors may provide a means of screening for the presence of compounds such as illegal growth promoters. Before such sensors can be utilised, it is necessary to understand which factors influence the response of an electrochemical sensor in bovine urine. The concentration range of protein (0.01–0.04%), uric acid (0.5–0.65 mM), xanthine (0.02–0.12 mM) and ascorbic acid (0.1–0.95 mM) in 26 individual urine samples were determined. Using p-aminophenol ( p-AP) as a model system, the electrochemical response increased by 5% in the presence of 6.0 mM uric acid, by 10% on the addition of 0.2 mM xanthine and by 22% in the presence of 1.0 mM ascorbic acid. Exposing urine to air and light for 75 min eliminated interference from ascorbic acid. Addition of Cu 2+ (10 μM) reduced the time required to 34 min. Binding of species such as growth promoters to proteins may be disrupted by the addition of 8-anilino-1-naphthalene sulphonic acid (ANS) to the urine samples. Addition of 10 μM ANS did not affect the limit of detection of p-AP. The pH of fresh bovine urine samples was monitored over the period 7 to 192 h after collection and ranged from 8.00 to 8.77. The pH of lyophilised urine samples ranged from 8.24 to 9.60. Amperometry was the most sensitive method among a range of electrochemical techniques in the detection of p-AP with a limit of detection (LOD) in urine of 1.0 μg mL −1 (10 μM) on a glassy carbon electrode.

Detection of illegal growth promoters in biological samples using receptor binding assays

In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time-consuming chromatographic methods of analysis or, sometimes, for screening purposes, on radio- or enzyme-immunoassays, all of which are often too specific to allow effective multi-analyte control. In order to develop rapid and inexpensive multi-analyte detection tests, we proposed the use of hormonal receptors as detection tools. The system described here (radio-receptor assays) is based on a direct binding assay of steroid hormones to their respective receptors. Human receptors to estrogens (hERα), androgens (hAR), progestagens (hPR) and glucocorticoids (hGR) have been produced by genetic engineering in bacteria or in eucaryotic cells. Binding analyses revealed that the obtained receptor proteins retained a high affinity for their corresponding native ligand. In addition, competition studies confirmed that each of the four receptors displays a specificity profile for a series of analogs in agreement with the literature. Finally, the stability of these recombinant receptors is sufficient to allow their use in test kits.

Recent developments in the use and abuse of growth promoters

During the last few years, control within the European Union (EU) for illegal growth promoters in cattle and pigs revealed only a limited number of positives. Analysis of illegal preparations, however, showed that steroids, often (esters of) natural hormones, and β-agonists are still used. Corticosteroids, controlled to a much lesser extent, seem to have become the most important group, while even thyreostats remain. Alarming information was obtained from specific investigations in which a large variety of products were found, some of which had never been reported to be misused in the field of growth promotion. For β-agonists and quinoxaline compounds, analogues of known compounds are synthesised. Other compounds are readily available as they are registered as growth promoters in some countries outside Europe or are allowed for specific veterinary purposes. Some classes of veterinary drugs are misused for their secondary pharmacological effects, e.g. benzodiazepines as feed intake enhancers and non-steroidal anti-inflammatory drugs (NSAIDs) as pale meat-making agents. Several non-traditional substances are suspected to be used in the field of breeding animals. This is the case for growth hormones (GHs) and all substances acting over this anabolic compound, as for instance, orally GH secretagogue. Moreover, ecdysteroids, which according to old Russian studies, have anabolic activity, are actually very easy to purchase on the Internet. Recent findings in different classes of growth promoters are discussed in detail.

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.

Multiresidue analysis of β 2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Various β 2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β 2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

Nortestosterone is not a naturally occurring compound in male cattle.

SummaryNortestosterone (β‐NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17α‐epimer (α‐NT). However, this also occurs naturally in pregnant cattle. It is not known whether α‐NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether α‐NT is naturally produced in this subset of the population. Bile samples from a total of 1,281 cattle (73 bulls and 1,208 steers) from 366 herds were collected at slaughter and initially screened by using a semi‐automated EIA with multi‐analyte immunoaffinity chromatography (IAC) clean‐up. Bile samples from a further 38 male cattle (10 bulls and 28 steers) were analysed by high‐resolution gas chromatography‐mass spectrometry (GC‐MS) with IAC pretreatment. Only samples containing more than 2 ng/ml α‐NT were subjected to GC‐MS. With 2 ng/ml α‐NT as a threshold for confirmatory testing, the false positive rate of the screening EIA was 1.8%. Bulls (n = 16) and steers (n = 179) from government farms (n = 2) and which were not treated with exogenous β‐NT, did not have measurable concentrations of α‐NT in their bile. Bulls (n = 35) and steers (n = 606) taken from herds (n = 204) which had no previous history of illegal growth promoter abuse also did not have α‐NT in their bile. Of 32 bulls and 451 steers of unknown treatment history sampled from herds (n = 160), 56 steers from 19 herds contained GC‐MS confirmed concentrations of α‐NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had β‐NT‐containing injection sites and five had residues of the β‐agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positivetanimals strongly suggested that exogenous 13‐NT had been administered at the presenting farm. It is concluded that α‐NT is not endogenous to this subset of the cattle population and that detection of this hormone in bile from bulls and steers constitutes evidence of abuse.

SOLID PHASE EXTRACTION FOR MULTI-RESIDUE ANALYSIS OF ANABOLIC STEROIDS AND RELATED SUBSTANCES FROM CALF URINE USING C18 AND ALUMINA COLUMNS

A solid phase extraction method for anabolic steroids and related substances in calf urine is reported, that is suitable as a screening method for illegal growth promoters. Two types of sorbent were used: a reversed phase C18 material and a polar alumina material. After overnight enzymatic deconjugation, the 5-mL sample was first brought on the C18 column. This column was washed with 55% methanol in water and was then eluted with 95% acetone in water. The extract was directly brought on the alumina column. The run-through and an additional elution with 95% acetone in water were collected. The final extracts were analysed with an HPLC-DAD method described previously. The steroids are separated on a Superspher RP-Select B column with a gradient mobile phase consisting of acetonitrile and water. The method was found to be suitable for at least 19 illegally used anabolic steroids, having recoveries ranging from 65–110% at a spiking level of 25 ng/mL. Detection limits ranged from 0.6–80 ng injected amount or 1–160 ng/mL urine.

Detection of residues using multivariate modelling of low-level GC-MS measurements

The chemometric analysis of low-level analytical data is hampered by the common presence of interfering compounds, by the frequent absence of measurement signals and by a non-constant measurement variability which is related to concentration level in a non-linear way. A model is presented to handle this type of data in the context of the practical problem of multivariate detection from gas chromatography/mass spectrometry (GC-MS) data. The model, based on log ratio modelling, is compared with previous approaches to parts of the problem. The basic idea behind the model is to define for the multivariate detection problem a null hypothesis for the values of log ratio measurements and to estimate variability as a function of total measured intensity. In practice it is often impossible to anticipate all kinds of interference which may occur. Therefore we propose to use expert assessments of the probability that certain expected peak ratios are generated by the analyte rather than by interferences. These expert assessments can then be used to define a proper null hypothesis for the multivariate detection test. The application of the model is illustrated for the detection of the illegal growth promoter clenbuterol in urine by selected ion-monitoring GC-MS.

Illegal growth promoters.

Illegal growth promoters.