Gallina, Guglielmo (2008) Marker urinari di trattamenti illeciti nel vitello: il residuo, i suoi metaboliti e nuovi parametri fisiologici. [Ph.D. thesis]
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In the EU the use of hormonal substances is prohibited in cattle fattening to avoid consumer exposure to unforeseeable risks from the intake of hormone residues and their metabolites. National Surveillance Plans for steroid abuse adopted the detection of the parent compound in urine and other sample matrices but, in spite of the progress in analytical techniques which allows to obtain very low detection limits for individual residues, this strategy may be unsuccessful; the use of natural hormones or of hormone precursors, the use of a combination of different active principles either at very low dosages or of unknown chemical structure are a few examples of procedures to escape the analytical controls. So effective surveillance and monitoring of the ban requires not only the availability of validated analysis methods for screening and confirmation of hormone residues in urine and other sample matrices but also knowledge about the absorption, biotransformation and excretion kinetics of illegally administered hormones, as well as endogenous levels of hormones in these sample matrices and also to develop complementary screening tests based on the biological action(s) of the substance(s) under investigation.
Aim of the thesis was to study the application of traditional analytical methods like LC/MS-MS , RIA and ELISA to detect to study the pattern of excretion of some urinary steroids in calves as marker of illicit treatments.
In the first part of this thesis is presented a preliminary assessment of physiological concentrations of testosterone (T) and epitestosterone (epiT) in urine of veal calves. Current methods to detect the misuse of natural androgens in cattle do not involve evaluation of urinary content of testosterone (T) because no validated physiological levels are set and threshold values cannot be fixed. In man urinary excretion of epiT occurs at similar or slightly lower concentration than that of T. Because of after exogenous T administration urinary epiT remains rather constant or decreases, while T increases, a threshold value of the ratio T/epiT = 6, in urine, was established as a limit for doping control in athletes. To evaluate whether urinary ratios could be useful for surveillance of exogenous hormone treatments also in calves urine samples were collected from control calves and from calves treated with oestradiol plus testosterone or oestradiol plus boldenone and urinary concentration of T and epiT was assessed by LC-MS/MS analysis. In all urine samples collected from control and treated animals the epiT concentrations were by far greater than T. EpiT may be considered one of the major urinary metabolites of T. Thus when the T/epiT ratio was calculated using the same approach adopted for doping control in man, no significant differences could be observed between treated and control calves. It is likely that the paramount difference between E and T concentrations masked the effect of treatment. Recent data, suggested that in calves repeated treatments with natural hormones could cause a time-course reduction of urinary E . Adopting the E/T ratio, the reduction of E urinary excretion can be confirmed in Testosterone treated calves (A) but still no evident in Boldenone treated ones.
The second part of this thesis deals with the identification of administation of a glucocorticoids like dexamethasone, (DXM) which is often illegally used as a growth promoters. To identify indirect biomarkers of illicit treatments, the urinary ratio between 6beta-hydroxycortisol (6beta-OHF) and cortisol (F), was measured in urines obtained from bulls experimentally treated per os and intramuscuarly (im) with different DXM dosages. Dexamethasone, given per os at low doses elicited an early and lasting significant reduction of 6beta-OHF/F. No significant variations were seen in urines from bulls given DXM intramuscularly. These results suggest 6beta-OHF/F as a rapid, non invasive, screening test for oral, low-dose, long-term corticosteroid treatment in cattle.
The last part regards the results of a study aiming to clarify the natural occurrence and the metabolism of boldenone in cattle.
The residue profiles of 17?-/17?-boldenone conjugated (17?/?-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of phytosterols and administered with 17?-Bol and ADD per os. The presence of conjugate 17?-Bol, ubiquitous and at higher concentrations in urine from control calves receiving the milk replacer with the greater amount of plant sterols support the theory that 17?-Bol is diet-related. In urine from treated animals the much higher17?-Bol levels were not modified by the production from diet precursors. The results confirmed the urinary excretion of 17?-Bol is due to exogenous administration of 17?-Bol. The discontinuous rate of elimination of both 17?- and 17?-Bol, despite the daily administration of 17?-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.
To identify metabolites as in vivo markers to support the thesis of exogenous administration, a further approach to the in vitro biotransformation of 17?-Bol and ADD was performed using different subcellular fractions obtained from both liver and kidney of untreated cattle. Metabolites obtained from incubated parent compounds were separated by liquid chromatography (HPLC) elution and successively identified by mass spectrometry (LC-MS/MS) detection. The bovine liver was the target tissue of the main metabolic reaction transforming 17?-Bol to ADD and viceversa. The analysis of the incubation extracts detected thepresence of polar hydroxylated (m/z 303) derivates of 17?-Bol, 17?-Bol and ADD. The results indicated that the urinary excretion profile in vivo of 6?-hydroxy-17?-Bol, 16?-hydroxy-17?-Bol and 6?-hydroxy-17?-Bol could be studied together with 17?- and 17?-BOLD as putative markers of 17?-Bol treatment in cattle.
Preliminary results of investigation regarding the presence of the hydroxylated metabolites in urine of calves treated with boldenone shown, even though additional studies are in due course, candidate 6?-hydroxy-17?-Bol as new potential marker of anabolic abuse of boldenone.
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