Investigations on the origin of prednisolone in cow urine

Prednisolone belongs to the corticosteroids that are molecules important in physiological protein, carbohydrate and fat metabolism and for their role in controlling inflammation. Besides naturally occurring corticosteroids (cortisol, cortisone), which are secreted by the adrenal cortex, a whole range of chemical synthetic analogs has been developed. Some of these synthetic corticosteroids (e.g. dexamethasone and prednisolone) have a legal use for therapeutic reasons only and maximum residue levels (MRLs) are established in some matrices as bovine liver, muscle, fat and milk. In urine no MRLs have been set for corticosteroids and then their presence at any concentration is not allowed. On the other hand corticosteroids are known to increase weight gain, to reduce the feed conversion ratio and to have a synergetic effect with other molecules like b-agonists. In the EU legislation the use of the corticosteroids as growth promoters in livestock breeding is prohibited. During 2008-2009 the analyses carried out as part of the Italian Residue Control Plan showed the presence of prednisolone in cow urine, especially in samples collected at the slaughterhouse. In all cases no therapeutic use was declared in advance by the farm veterinarians. We therefore hypothesised that the stress due, for example, to the handling of the animals before slaughter caused prednisolone and prednisone to increase to levels that could be detected using current analytical methods. The stress also increased urinary levels of cortisol and cortisone in cattle. Accordingly, in a first study we measured the urinary concentrations of cortisol, cortisone, prednisolone and prednisone in dairy cows before and after the pharmacological induction of stress by intramuscular administration of tetracosactide hexaacetate (an ACTH synthetic analogue). The urine of each animal was also analysed after slaughter. The results published [Steroids 76 (2011) 104–110] suggested that prednisolone could be endogenously produced. In the present trial, 52 lactating cows housed in 7 different Lombardy farms were studied. Urine samples were collected at the farm and immediately after the slaughter. The results obtained confirmed that prednisolone could be endogenously produced and furthermore its presence in bovine urine seems to be strongly related to a state of stress in the animals (at farm and slaughterhouse). Conclusions: The possibility that prednisolone can be endogenously produced by dairy cows was confirmed. The presence of endogenous prednisolone in bovine urine seems to be strongly related to a state of stress of the animals (at farm or slaughterhouse). Presently it is hard to set an undoubtedly endogenous prednisolone urinary threshold concentration. This is due to the quite high variability of the glucocorticoid levels, related to the welfare conditions of the animal and to the circadian rhythm of ACTH release. A parameter, i.e. the presence of certain metabolites or the ratio cortisol (or cortisone) to prednisolone, should be therefore studied and explained to allow the official control organizations to take the most possible accurate decisions about the cause of the prednisolone presence in urine (either endogenous or following a treatment). Results and discussion The liquid chromatography-mass spectrometry (LC-MS/MS) method used was fully validated, in agreement with Commission Decision 2002/657/EC criteria, for the confirmation of 11 corticosteroids (triamcinolone, prednisolone, prednisone, cortisol, cortisone, methylprednisolone, betamethasone, dexamethasone, flumethasone, beclomethasone and triamcinolone acetonide). It permitted the detection and quantification of all the analytes above 0.4 μg/L. The urine samples were also analyzed for the presence of microbiological contamination and found negative. An overview of the data from LC-MS/MS analyses performed in this study are reported in Table 1 and Table 2. Prednisone and any other corticosteroids besides prednisolone, cortisol and cortisone were never detected. The results presented in Table 1, with regard to prednisolone, show that animals “negative” at farm both 24 and 2 hours before the transport to the slaughterhouse became “positive” when sampled at slaughterhouse; this fact can be explained only assuming that endogenously prednisolone can be produced by the animal. Furthermore no animals found “positive” at farm were “negative” at slaughterhouse. Table 2 shows that the frequency of prednisolone detection at slaughterhouse was much higher than in farm, and furthermore that the prednisolone concentrations found at slaughterhouse and farm were quite the same. The highest concentrations found were 1.7 μg/L at farm and 2.2 μg/L at slaughterhouse. Cortisol and cortisone were always detected, but the measured concentrations, were extremely variable, probably due to their circadian rhythm. However both at farm and slaughterhouse, when prednisolone was detected, the cortisol and cortisone concentrations were on average higher than their levels when prednisolone was not detected. This can be explained assuming that the prednisolone detection was related to a state of stress of the animals.