Effects of truck transportation and slaughtering on the occurrence of prednisolone and its metabolites in cow urine, liver, and adrenal glands.

BACKGROUND: The recognition of illegal administration of synthetic corticosteroids in animal husbandry has been recently challenged by the case of prednisolone, whose occasional presence in the urine of bovines under strong stressful conditions was attributed to endogenous biosynthesis, not to exogenous administration. The study of the natural stress sources possibly inducing endogenous prednisolone production represents a stimulating investigation subject. The biochemical effects of transportation and slaughtering were verified in untreated cows by studying the possible occurrence of prednisolone and its metabolites in urine, liver and adrenal glands, and the cortisol/cortisone quantification. RESULTS: Cortisol, cortisone, prednisolone and its metabolites were measured in urine, collected at farm under natural micturition and then at the slaughterhouse. The study was performed on 15 untreated cows reared in different farms at the end of their productive cycle. 2-3 days after the first urine collection, the animals were transported by trucks to the abattoir, slaughtered, and subjected to a second urine sampling from the bladder. Specimens of liver and adrenal gland were also collected and analysed by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated method. The stressful conditions of transportation and slaughtering proved to increase considerably the urinary levels of cortisol and cortisone as compared to those collected at farm. Prednisolone was detected in the urine collected at the slaughterhouse of two cows only, at a concentration level (≈0.6 µg L- 1) largely below the official cut off (5.0 µg L- 1) established to avoid false non-compliances. These two animals exhibited the highest urinary cortisol levels of the series. Prednisolone and prednisone were also detected in the adrenal glands of a different cow. Prednisolone metabolites were not detected in any urine, liver, and adrenal gland sample. CONCLUSION: Within the constraints of the condition adopted, this study confirms the sporadic presence of prednisolone traces (2 samples out of 15) and the consistently increased concentration of cortisone and cortisol in the urines collected from cows subjected to truck transportation and subsequent slaughtering. No prednisolone metabolites were detected in any liver and adrenal gland samples, nor in urine specimens, unlike what was previously reported for cows artificially stressed by pharmacological treatment.

GR CALUX assay detects synthetic glucocorticoids in calf urine: a validation study.

Member States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the ß error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL-1 concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL-1 for flumethasone, dexamethasone, betamethasone; < 4 ng mL-1 for methylprednisolone; 5 ng mL-1 for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity.

Determination of endogenous and exogenous corticosteroids in bovine urine and effect of fighting stress during the "Batailles des Reines" on their biosynthesis.

Natural corticosteroids include two families of substances: mineralocorticoids and glucocorticoids. Several drugs of similar structure and biological activity have been synthesized and are currently used in the clinical practice. Beside legal pharmacological treatments, these drugs have been consistently misused in animal breeding. One of the most abused corticosteroids is prednisolone. For many years, prednisolone has been considered of exclusive synthetic origin, but nowadays a debate about its possible endogenous production is under way. Several studies have been addressed to ascertain the potential relationship between stressful conditions, such as transportation and slaughtering, and endogenous production of prednisolone. In order to verify further the effect of stressful conditions, our laboratory analysed urine samples collected from the cows participating to the "Batailles des Reines" (a traditional contest based on ritual and spontaneous fights of pregnant cows), to verify if an endogenous prednisolone production may occur in these animals. We developed and validated a LC-MS/MS method for the simultaneous determination of cortisol, cortisone, prednisolone and five of its metabolites. The method was applied to the analysis of urine samples collected from "Batailles des Reines" competitions in 2012 and 2013. All these samples had been previously analysed within an anti-doping control program and tested compliant to all screenings.

Effect-based detection of synthetic glucocorticoids in bovine urine.

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose-response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay's ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ≫ dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA.

Profile of the urinary excretion of prednisolone and its metabolites in finishing bulls and cows treated with a therapeutic schedule.

BACKGROUND: Prednisolone was one of the first glucocorticoids to be synthesised, but it is still widely applied to cattle. Illegal uses of prednisolone include its uses for masking a number of diseases before animal sale and, at lower dosages for extended periods of time, for the improvement of feed efficiency and carcass characteristics. Since occasional presence of prednisolone has been detected at trace level in urine samples from untreated cattle, the Italian Ministry of Health introduced a provisional limit of 5 ng/mL to avoid false non-compliances. However, this limit proved ineffective in disclosing prednisolone misuse as a growth-promoter. In the present study, prednisolone acetate was administered to finishing bulls and cows according to a therapeutic protocol (2 × 0.4-0.5 mg/kg bw i.m. at 48 h interval) to further verify the practical impact of this cut-off limit and develop sound strategies to distinguish between exogenous administration and endogenous production. Urinary prednisolone, prednisone, 20ß-dihydroprednisolone, 20α-dihydroprednisolone, 20ß-dihydroprednisone, 6ß-hydroxyprednisolone, cortisol, and cortisone were determined using a validated LC/MS-MS method. RESULTS: The urinary excretion profile showed the simultaneous presence of prednisolone, 20ß-dihydroprednisolone, and prednisone, the latter at lower concentrations, up to 33 days after the first dosing. Higher analyte levels were detected in bulls even after correction for dilution in the urine. Prednisolone concentrations below 5 ng/ml were determined in half of the samples collected at 19 days, and in all the samples obtained 26 and 33 days after the first administration. No measurable concentrations of prednisolone or its metabolites were found in the samples collected before the treatment, while cortisol and cortisone levels lower than the respective LOQs were observed upon treatment. CONCLUSIONS: The present study confirms the criticism of the coarse quantitative approach currently adopted to ascertain illegal prednisolone administration in cattle. As previously shown for growth-promoting treatments of meat cattle, the simultaneous determination of urinary prednisolone, prednisone, 20ß-dihydroprednisolone, along with cortisol and cortisone, may represent a more reliable approach to confirm the exogenous origin of prednisolone. Such a strategy would facilitate unequivocal detection of animals treated with prednisolone acetate using a therapeutical protocol, even 3 to 4 weeks after the treatment.

Fit-for-purpose in veterinary drug residue analysis: development and validation of an LC-MS/MS method for the screening of thirty illicit drugs in bovine urine.

A selective and sensitive method for screening 31 analytes (nine corticosteroids, eight ß-agonists, seven anabolic steroids, six promazines and zeranol) in bovine urine was validated according to 2002/657/EC guidelines. Upon optimization of sample treatment conditions, the extraction was performed by diethylether at pH 9, after deconjugation. Extraction yields (R%) proved higher than 70% for 19 analytes, 50

Effects of low-dose dexamethasone and prednisolone long term administration in beef calf: chemical and morphological investigation.

An analytical, pharmacokinetic and histopathologic investigation was conducted by two experimental trials on beef cattle in order to determine fate and effects of dexamethasone and prednisolone, administered to distinct cattle groups at low dosage for long periods of time. In trial 1, eighteen Charolaise beef cattle, male, 17-22-months-old, were divided in three groups: to group A (n=6) dexamethasone-21-sodium-phosphate 0.7 mg day(-1) per os for 40 days was administered; group B (n=6) was orally treated with prednisolone 15 mg day(-1) for 30 days, while group C (n=6) served as negative control. Urine was collected at days 0, 7, 15, 25 and 47 from groups A and C, and at days 0, 8, 18 and 42 from group B. In trial 2, sixteen Friesian cattle, male, 10-17-months-old, were randomly divided into two groups: group D (n=8) was administered prednisolone 30 mg day(-1) per os for 35 days, while group K (n=8) served as control. In both trials, the animals were slaughtered after a 6-days drug withdrawal and thymus and livers were collected and properly stored until the analysis was performed. Quantitative determinations of dexamethasone, prednisolone and its main metabolite, prednisone, in urine and liver samples were conducted by HPLC-MS/MS, after the analytical procedure was optimized and fully validated. The method validation included the assessment of specificity, linearity, precision, trueness, robustness, CC(α) and CC(ß) values. By a morphological point of view, severe atrophy of thymus parenchyma was observed in group A, together with a significant (P<0.005) reduction of the mean thymus weight (217±94 g), while group B (646±215 g) presented normal thymus features and weights (group C, 415±116 g). Accordingly, no differences were found in trial 2 for groups D (727±275g) and K (642±173 g). Average dexamethasone concentrations in group A urine samples ranged from 1.4 to 3.0 µg L(-1) during the treatment, while no residue was detected in the urine samples collected 6-7 days after the end of the treatment. Low amounts of dexamethasone (<1 µg L(-1)) were detected in liver samples of group A. All average prednisolone concentrations in group B urine samples (sum of conjugate and free form) turned out to be below 1.0 µg L(-1) during the treatment, despite the much higher concentration administered (15-30 mg day(-1)) with respect to dexamethasone in group A (0.7 mg day(-1)). No prednisolone residues were found in the urine and liver samples taken at the slaughterhouse. The absence of any prednisolone residue in the urine samples of control group animals supports the theory that the origin of this molecule is fundamentally exogenous, at least for this cattle category maintained under unstressing conditions. Remarkable findings are represented by the absence of thymus atrophy in the prednisolone treated animals and the extremely low residue concentrations found in urine during the treatment. Both findings reveal that the detection of illegal growth-promoting treatments with this drug is difficult.

Study of dexamethasone urinary excretion profile in cattle by LC-MS/MS: comparison between therapeutic and growth-promoting administration.

Dexamethasone is a potent synthetic corticosteroid widely employed as a therapeutic agent in cattle. Besides this legal use, corticosteroids are also administered at low dosages as growth-promoters either alone or in combination with other steroids or with beta-agonists. For this reason, appropriate control plans are established to survey corticosteroid misuse, using liver or urine as biological matrices. Since few data are available about the kinetics of dexamethasone excretion in meat cattle, an experimental study was designed to assess the drug residue levels in urines following either a therapeutic (60 microg of dexamethasone sodium phosphate/kg b.w., for three consecutive days) or a growth-promoting schedule (0.7 or 1.4 mg of dexamethasone sodium phosphate per capita/day for 60 days). The urinary elimination of dexamethasone, which was predominantly excreted in the unmodified form, was determined by high-performance liquid chromatography/tandem mass spectrometry at different time intervals, i.e. during the treatments and after appropriate withdrawal times. Our findings confirm the high and rapid rate of dexamethasone urinary excretion irrespective of the nature of the treatment, and provide useful reference values that can be conveniently employed for forensic purposes.