The impact of stress on the prevalence of prednisolone in bovine urine: A metabolic fingerprinting approach

Recent studies support the hypothesis that the glucocorticoid prednisolone can be formed from cortisol under influence of stress. To evaluate this hypothesis, urine samples of supposedly non-stressed bovines (at the farm) and bovines subjected to two different forms of stress, i.e. upon slaughter (natural stress) or following administration of a synthetic analog of the adrenocorticotropic hormone (pharmacologically-induced stress) were analysed, and their urinary cortisol and prednisolone levels evaluated. At the farm, none of the examined samples exhibited urinary prednisolone levels higher than the CCα (0.09 μg L(-1)). Upon slaughter or following synthetically induced stress, significantly positive correlations between cortisol and prednisolone could be demonstrated, 0.52 and 0.69, respectively. Of all prednisolone-positive urine samples (n=84), only one showed a prednisolone levels (i.e. 6.45 μg L(-1)) above the threshold level of 5 μg L(-1) suggested by the European Reference Laboratories. Subsequently, an untargeted analysis was performed (metabolic fingerprinting) to characterize the urinary metabolite patterns related to the three different cattle groups. In this context, multivariate statistics assigned a total of 169 differentiating metabolites as playing a key role in the urinary pattern in response to stress. Three of these ions were defined as steroids using an in-house created database. As a result, the metabolic fingerprinting approach proved to be a powerful tool to classify unknown bovine urine samples that tested positive for prednisolone, while providing information about the stress status of the animal.