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THE DOUBLE BOND IN Δ1-2 POSITION OF STEROIDS: A MATTER OF CONTROVERSY IN THE CONTROL OF ILLICIT TREATMENTS OF FARM ANIMALS
Full text linkTwo tight correlated topics were dealt in this thesis: - Bovine urine can result positive for boldenone, an androgenic steroid with the structure of testosterone with a double bond in Δ1-2 position of ring A, and androstadienedione, its 17-oxidized form, in consequence of faecal contamination, mainly through a high neo production in faeces-contaminated urine. A simple in vitro method was developed – incubation of faecal matter suspended in 0.9% saline – to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Androstenedione, androstadienedione, alfa and beta boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversions of steroids were observed, although 17 beta hydroxy steroids had lower stability if compared with 17 alfa hydroxy and 17-keto steroids. This simple in vitro system proved to be an effective way of studying hormone transformations in faeces and in faeces-contaminated urine. An analogous method, i.e. the direct injection, for LC/MS-MS analysis, of diluted equine urine, without prior extraction, was carried out to evaluate the stability of phase II metabolites of boldenone, testosterone and epitestosterone (glucuronides and sulphates) in intentionally poorly stored urine. Both 17 beta and 17 alfa-glucuronide steroids hydrolyzed to their correspondent hydroxysteroids, but only the 17 beta epimers oxidized to the keto forms. The sulphate compounds showed a complete stability. The transformations were temperature dependent: faster at room temperature than at 4°C, while did not occur in frozen urines. This indicated that proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of androgen steroids. - After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. The problem of modifications in biological fluids was therefore evaluated also regarding a possible transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces. The transformation of the II phase metabolite cortisol glucuronide was studied, too. The method consisted once more of incubation of bovine faecal matter suspended in 0.9% saline spiked with cortisol, cortisone and cortisol glucuronide (400 ng/ml). The deconjugation of the conjugated form the and the dehydrogenation in ring A occurred. It was so concluded that also urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone. The successive step was to assess the transformation of cortisol and cortisone in urine collected from the bladder at the slaughterhouse (i.e. without faecal contamination) and incubated at 37°C for 24 hours. The influence of deconjugation with beta-glucuronidase from Helix pomatia was assessed, too. Only urine where prednisolone and prednisone were absent and cortisol was higher than 80 ng/mL were used. In this study a liquid-liquid extraction with a mixture of tert-Buthylmethylether:Ethylacetate (80:20) was performed, to concentrate the sample, so raising the sensitivity of the method. The analyses were carried out by LC MS3. It was demonstrated that a relatively high temperature (e.g. due to inappropriate storage conditions or to enzymatic deconjugation), could give results that do not represent the effective presence and concentration of the abovementioned corticosteroids at the moment of the sample collection. However, because of the stressful conditions of the slaughterhouse, the by-production of prednisolone was supposed, together with the increase of cortisol and cortisone. The urines of three dairy cows were therefore collected and analyzed before and after a pharmacological induced stress (intra muscular administration of tetracosactide hexaacetate, an adrenocorticotropic hormone analogue), and before and after slaughter. Prednisolone was occasionally detected in the non-stressful state, but was consistently found in the urine of stressed cows (from 1.01 to 4.08 ng/mL), together with the raise in concentration of urinary cortisol and cortisone, so indicating a possible endogenous production of prednisolone in stressful situations
Recettori alfa adrenergici postsinaptici responsabili di vasocostrizione dell’arteria digitale comune isolata di cavallo
No full text[Nell'arteria digitale comune del cavallo sono stati studiati, in vitro, gli effetti degli agonisti e degli antagonisti alfa inf (1)- e alfa inf (2)-adrenergici, allo scopo di caratterizzare dal punto di vista funzionale gli effetti degli stimoli determinati dai recettori alfa-adrenergici postsinaptici. Contrazioni dipendenti dalla concentrazione determinate dalla fenilefrina (alfa inf (1)-agonista) venivano contrastate competitivamente sia dalla prazosina, sia dalla yohimbina, con affinita' diverse. La detomidina e l'UK14304 (alfa inf (2)-agonisti) provocavano effetti "tutto o nulla". Questi risultati fanno ipotizzare un coinvolgimento predominante degli alfa inf (1)-recettori nel controllo della contrazione del substrato mentre, al momento, non spiegano il ruolo della stimolazione degli alfa inf (1)-adrenorecettori]The effects of alpha sub (1)- and alpha sub (2)-adrenoceptor agonists and antagonists were studied in vitro on equine common digital artery to characterize functionally the effects of postjunctional alpha sub (1)- and alpha sub (2)-adrenoceptors stimulations. Concentration-dependent phenilephrine (alpha sub (1)-agonist) contractions were competitively antagonized both by prazosin and yohimbine with different affinities. Detomidine and UK14304 (alpha sub (2)-agonists) induced all or none effects. These results suggest a predominant involvement of alpha sub(1)-adrenoceptors in the contractile control of the substrate while, up to date, do not explain the role of alpha sub (2)-adrenoceptors stimulatio
Presence of residues in food of animal origin and related risk
No full textThe risk related to the presence of residues in food of animal origin has to be carefully evaluated and it is important to distinguish between the residues of veterinary drugs and environmental contaminants. The first are easily monitored and only difficulty and occasionally exceed MRL concentrations, whereas the second ones can have prolonged and high-level exposures. The Safety Margins of environmental contaminants are thus lower, and the risk increases for high level consumers. It is therefore important to ovoid transfer of pollutants to food of animal origin, especially when local contamination occur
Identificazione dei ß-recettori coinvolti nella lipolisi nel tessuto adiposo sottocutaneo di vitello
No full textAdrenergic regulation of vascular smooth muscle tone in calf digital artery
No full textRadioligand binding studies and functional assays on isolated smooth muscle preparations were performed in order to obtain a biochemical and functional characterization of the beta-adrenoceptor (beta-AR) subtypes involved in regulation of the smooth muscle relaxation of the calf's common digital artery. The results indicate that the common digital artery possesses two beta-AR populations (40% beta(1) and 60% beta(2)) and the beta(2)-subtype appears to predominate as far as function is concerned. Only the beta(2)-AR agonists clenbuterol and fenoterol caused dose-related relaxant effects, antagonized by propranolol, when tested in preparations precontracted both with PGF(2alpha) (1.4 x 10(-5) m) and noradrenaline (1.2 x 10(-6) m). In noradrenaline precontracted preparations the beta(1)-AR selective agonists dobutamine and xamoterol caused vasodilation which was not antagonized by (+/-)propranolol. While the functional relaxant effects of dobutamine may be attributed to its potent competitive alpha-AR blocking activity, further investigations are required to explain the effect of xamoterol. The vasodilator effect of (+/-)isoproterenol was irregular. The recorded contractile effects, mainly at dosages greater than 10(-6) m, suggest the loss of drug selectivity for beta-AR and alpha-AR activation. Indirect evidence indicates that the alpha-adrenoceptor (alpha-AR) population in this tissue which produces a strong contraction is functionally dominant over the beta-AR, suggesting limited therapeutic benefit for beta-AR drugs to control blood flow disorders in the calf's distal limb
Ruolo dei recettori tachichininici NK2 nella contrazione della muscolatura liscia circolare dell’ileo di cavallo
No full textIn vitro transformation of boldenone and related steroids in veal faeces : preliminary results
No full textPrednisolone and prednisone neo-formation in bovine urine after sampling
No full textThe rise in the frequency of detecting prednisolone in bovine urine from northern Italy has come into focus of attention in recent
years. The possibility that neo-formation of prednisolone or that prednisone may occur in urine after collection of samples was
therefore investigated. Cow urine collected for official routine controls in Lombardy containing more than 80 ng/ml cortisol,
and prednisolone and prednisone below the decision limit (CCa) of the method (0.4 and 0.5 ng/ml, respectively) was used.
The C1–2 dehydrogenation of naturally present cortisol and cortisone was checked by incubating urine, both contaminated and
uncontaminated with faeces, at 378C and by collecting samples at 0, 1, 2, 4, 6 and 24 h. The influence of Helix pomatia juice
was also investigated in order to determine whether deconjugation could influence the reliability of the results. All samples were
analysed by HPLC-MS3 for the presence of cortisol, cortisone, prednisolone and prednisone in negative electrospray ionisation
mode, utilising the consecutive reaction monitoring of product ions derived from the formate molecular adduct ([M1HCOO]–).
The observed neo-formation of prednisolone shows that inappropriate temperatures in sample storage and processing can result
in an incorrect accusation of non-compliance. The faecal contamination of urine, performed with the aim to mimic a collection
conducted without the necessary care, moreover, evoked a high increase in prednisolone concentration in two out of seven
animals. Moreover, H. pomatia juice had no significant effect on the prednisolone concentration, indicating that this
corticosteroid is present in its free form in cow urine
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