Profiling of metabolomic changes induced by testosterone esters in pig plasma and urine
The use of hormones as growth promoters for fattening purposes in livestock has been banned in the European Union since 1988 by Council Directive 93/22/EC. However, this banned substances are still reported within the framework of European monitoring residue plans. In order to improve reliability of the detection methods, metabolomics approaches to non-targeted screening for detection of anabolic practices using the naturaly steroid hormones or new synthetic growth promoters have been designed recently [1]. In this study, metabolic fingerprinting to discriminate between pigs treated with 17β-testosteron esters and control animals has been investigated. Twelve 90 day-old pigs were randomly separated into test (8 animals) and control (4 animals) groups. Animals from the test group were treated with i.m. administration of the hormonal preparation (testosterone propionate, testosterone phenylpropionate, testosterone isocapronate, testosterone decanoate; Sustanon 250, N.V. Organon, CZ Reg.56/357/91-C). Urine samples were collected in both groups from 14 to 90 days after treatment. Plasma samples were collected from day 1 to day 90. All pigs were weighed every week within the experiment. Samples were filtered on centrifugal devices to remove proteins and obtained filtrates were mixed with internal standard (testosterone-D3 in methanol, Sigma-Aldrich). Each sample was injected into chromatographic system using an Accera 1200 Series on a Hypersil Gold C18 column for separation. LC-HRMS metabolomic fingerprints were acquired on Q-Exactive mass spectrometer (Thermo Fisher Scientific) in ESI+ mode. Full scan mass spectra were acquired from 80 to 800 m/z using a mass resolution of 70.000 FWHM in centroid mode. Raw data were processed by SIEVE and XCMS software. PCA and OPLS-DA were carried out using SIEVE, XCMS and SIMCA-P+ statistic software. The chromatographic analysis of the fingerprints generated 968 ions and 692 ions for plasma and urine, respectively. Statistical evaluation selected only significant peaks (p-value), 374 and 264 peaks were extracted for plasma and urine samples, respectively. Multivariate statistical analysis showed significant metabolic differences between test and control groups on day 28 after aplication of the testosterone hormonal preparation. However, no significant differences between treated and control group were found after day 42. Moreover, the urine samples were examined by conventional target LC-MS/MS. In this case, no testosterone residues were detected after day 14. The study showed also higher growth performance (p<0.05) in treated pigs in comparison to control animals, demonstrating anabolic effect of testosterone esters. The work is going on, additional experimental animals will be used to confirm the fingerprinting data to the control of banned testosterone preparation in pigs.
