Investigation on the origin of prednisolone in urine and adrenal glands of cows

Abstract

Prednisolone is a steroid belonging to the corticosteroid group. The results obtained in the application of the 2008 and 2009 Italian Residue Control Plans show the frequent detection of prednisolone traces in cow's urine. Since most of the positive samples were detected at the slaughterhouse, the researchers hypothesised that, together with an increase of cortisol concentration, traces of prednisolone could be produced endogenously during stressful situations due to transport and handling before slaughter. In the present trial, 52 lactating cows housed in seven different farms in Lombardy, Italy, were studied. Urine samples were collected at the farm (after urethral catheterisation) and immediately after slaughter (from urinary bladder) together with 40 adrenal gland samples belonging to the same animals. All the samples were analysed for the determination of prednisolone and cortisol by LC/MS(n). The results demonstrated that prednisolone can be endogenously produced in dairy cows and, furthermore, its endogenous presence in bovine urine seems to be strongly related to a state of stress in the animals (at the farm and at the slaughterhouse). The data from adrenal glands do not, however, clarify if the endogenous production occurs, partially or totally, in this organ.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment

“…Furthermore, considerable impact could be attributed to a different sampling schedule, since the last time point in our previous experiment was at slaughter, which intensified the extremely stressful situation. As was mentioned above, heavy stress conditions apparently favour endogenous PL production that is usually accompanied by an increase in urinary concentrations of F and E (Bertocchi et al 2013;Ferranti et al 2013), while exogenous administration of PL is likely to reduce the production of these main endogenous glucocorticoids. Our results confirm this hypothesis and are enriched with monitoring of the levels of F and E A-ring-reduced metabolites.…”

Pseudoendogenous origin of prednisolone in pigs from the food chain

“…The sample size calculation was made according to Bottarelli and Ostianello (2011) using the following equation: n = Z 2 Â P 1 À P ð Þ ½ =D 2 , where n is the sample size, Z is the Student's t value (1.96, when the level of significance is 5%), P the expected prevalence and D the required precision. At the time the experimental protocol was designed, we had no data on the frequency of prednisolone detection in pig urine, but only from bovines (cows at slaughter = 71% positive; Bertocchi et al 2013) and race horses (78.5%; Fidani et al 2012). Based on these frequencies, on their difference between cows and horses, and because the pigs, i.e.…”

“…Sample preparation was conducted as previously reported in Bertocchi et al (2013). A 5 g portion of the adrenal gland was transferred to a 50 ml tube and spiked to a concentration of 10 ng ml −1 with the internal standard prednisolone-d6.…”

“…Confirmation of the presence of prednisolone in the adrenal glands was made by LC-MS 3 on 8 samples already analysed by LC-MS 2 . The analysis, already used for bovine adrenal glands by Bertocchi et al (2013) was performed with an ion trap in the negative ESI mode. The results, performed only through qualitative determination, fully confirmed those reported in this work.…”

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

“…The usual nonsterile collection or transportation conditions and the presence of normal or pathogenic microbial flora contamination are all favorable circumstances that are able to change the composition of the urine specimens. Bacterial activity may lead to an increase or depletion of endogenous steroids or even to the hydrolysis of conjugated metabolites, altering the specific steroid profile parameters [11][12][13][14] Among the in situ bacterial reactions on endogenous steroids, 19-demethylation and Δ1-steroid-dehydrogenation, which lead to the formation of 19-norandrosterone (19-NA) and boldenone, respectively, have been extensively reported in the literature 15,16 The uncommon presence of prednisone and prednisolone in human urine may be attributed to the activity of Δ 1 -SDH on steroids (respectively cortisone and cortisol) physiologically excreted into urine [17][18][19][20] To discriminate the endogenous or exogenous origin of prednisone and prednisolone, WADA established the GC-C-IRMS analysis as a recommended confirmation at concentrations between the reporting level of 30 ng/mL and 60 ng/mL. The aim of this work was to determine the origin of prednisone and prednisolone by GC-C-IRMS, developing and validating a specific method in accordance with the WADA Technical Document TD2019IRMS 21 and the ISO17025 requirements.…”