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Metabolomics

, 13:77 | Cite as

Serum-based metabolomics characterization of pigs treated with ractopamine

  • Tao Peng
  • Anne-Lise Royer
  • Yann Guitton
  • Bruno Le Bizec
  • Gaud Dervilly-PinelEmail author
Original Article

Abstract

Introduction

Ractopamine, a β-agonist used as growth promoter in livestock, is with great controversy, and it has been forbidden in most countries worldwide. However, due to economic benefits, the possibility of widespread abuse of ractopamine still exists. “Omics” strategies, based on the observation of physiological perturbations, are promising approaches to tackle drug misuse in breeding animals.

Objectives

A study was performed to determine if serum-metabolomics could be used to establish a predictive tool for identifying ractopamine misuse in pigs.

Methods

Our aim was to set up a high performance liquid chromatography—high resolution mass spectrometry based metabolomics workflow for screening pig serum for ractopamine administration. Therefore, an untargeted metabolomics approach was developed to characterize and compare serum metabolic profiles from control and treated pigs. Two different extraction strategies were investigated, and the results showed that the combination of methanol extraction and methanol–water extraction protocols significantly improve the metabolites coverage. A two-level data analysis using univariate and multivariate statistical analyses was carried out to establish descriptive and predictive models.

Results

The discrimination of treated animals from control animals could be achieved. A number of candidate biomarkers that contributed the most in the observed discrimination could be listed.

Conclusion

This research indicates that metabolomics approach can be considered as a powerful strategy to highlight biomarkers related to ractopamine treatment in pig which may subsequently be implemented as screening strategy to predict for such illicit practices.

Keywords

β-Agonist Livestock Untargeted HPLC–HRMS Biomarker Omics 

Notes

Compliance with ethical standards

Compliance with animal studies and ethical standards

The animal study was approved by the national Ethical Committee (n°6) under agreement 2,015,092,516,084,715 / APAFIS 1914 (CRIP-2015-054).

Conflict of interest

The authors declare no conflicts of interest.

Compliance with ethical requirements

We confirm that this manuscript has not been published elsewhere and is not under consideration in another journal. All authors have approved the version of this manuscript and agree with its submission to Metabolomics.

Supplementary material

11306_2017_1212_MOESM1_ESM.docx (1 mb)
Supplementary material 1 (DOCX 1049 KB)

References

  1. Anderson, D. B., Moody, D. E., & Hancock, D. L. (2009). Beta adrenergic agonists. In W. G. Pond & A. W. Bell (Eds.), Encyclopedia of animal science (pp. 104–107). New York: Marcel Dekker, Inc.Google Scholar
  2. Bird, S. S., Marur, V. R., Sniatynski, M. J., Greenberg, H. K., & Kristal, B. S. (2011). Serum lipidomics profiling using LC-MS and high energy collisional dissociation fragmentation: Focus on triglyceride detection and characterization. Analytical Chemistry, 83, 6648–6657.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bottemiller H. (2012). Dispute over drug in feed limiting US meat exports. Retrieved from Sept 15, 2016 from https://thefern.org/2012/01/dispute-over-drug-in-feed-limiting-u-s-meat-exports/.
  4. Bottemiller H. (2013). Escalating trade dispute, Russia bans Turkey over ractopamine residues. Retrieved Sept 15, 2016 from http://www.foodsafetynews.com/2013/02/escalating-trade-dispute-russia-bans-turkey-over-ractopamine-residues/#.V7cJ_fmep8o.
  5. Boyard-Kieken, F., Dervilly-Pinel, G., Garcia, P., Paris, A. C., Popot, M. A., Le Bizec, B., et al. (2011). Comparison of different liquid chromatography stationary phases in LC-HRMS metabolomics for the detection of recombinant growth hormone doping control. Journal of Separation Science, 34, 3493–3501.CrossRefPubMedGoogle Scholar
  6. Catalano, D., Odore, R., Amedeo, S., Bellino, C., Biasibetti, E., & Miniscalco, B. (2012). Physiopathological changes related to the use of ractopamine in swine: Clinical and pathological investigations. Livestock Science, 144, 74–81.CrossRefGoogle Scholar
  7. Commission Decision 2002/657/EC. (2002). Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities, L221.Google Scholar
  8. Council Directive 96/22/EC. (1996). Concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of beta-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Official Journal of European Communities, L125.Google Scholar
  9. Council Directive 96/23/EC. (1996). On measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC. Official Journal of European Communities, L125.Google Scholar
  10. Courant, F., Antignac, J. P., Dervilly-Pinel, G., Le Bizec, B. (2014). Basics of mass spectrometry based metabolomics. Proteomics, 14, 2369–2388.CrossRefPubMedGoogle Scholar
  11. Courant, F., Dervilly-Pinel, G., Bichon, E., Monteau, F., Antignac, J. P., Le Bizec, B. (2009). Development of a metabolomic approach based on liquid chromatography high resolution mass spectrometry to screen for clenbuterol abuse in calves. The Analyst, 134, 1637–1646.CrossRefPubMedGoogle Scholar
  12. Courant, F., Royer, A. L., Chereau, S., Morvan, M., Monteau, F., Antignac, J. P., et al. (2012). Implementation of a semi-automated strategy for the annotation of metabolomic fingerprints generated by liquid chromatography-high resolution mass spectrometry from biological samples. The Analyst, 137, 4958–4967.CrossRefPubMedGoogle Scholar
  13. Dervilly-Pinel, G., Chereau, S., Cesbron, N., Monteau, F., Le Bizec, B. (2015). LC–HRMS based metabolomics screening model to detect various β-agonists treatments in bovines. Metabolomics, 11, 403–411.CrossRefGoogle Scholar
  14. Dervilly-Pinel, G., Courant, F., Chereau, S., Royer, A. L., Boyard-Kieken, F., Antignac, J., et al. (2012). Metabolomics in food analysis: Application to the control of forbidden substances. Drug Testing and Analysis, 4, 59–69.CrossRefPubMedGoogle Scholar
  15. Dervilly-Pinel, G., Weigel, S., Lommen, A., Chereau, S., Rambaud, L., Essers, M., et al. (2011). Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves. Analytica Chimica Acta, 700, 144–154.CrossRefPubMedGoogle Scholar
  16. Dunn, W. B., Broadhurst, D., Begley, P., Zelena, E., Francis-McIntyre, S., Anderson, N., et al. (2011). Procedures for large scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature Protocols, 6, 1060–1083.CrossRefPubMedGoogle Scholar
  17. Dyer, E. G., & Bligh, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917.CrossRefPubMedGoogle Scholar
  18. EFSA. (2009). Scientific opinion of the panel on additives and products or substances used in Animal Feed (FEEDAP) on a request from the European Commission on the safety evaluation of ractopamine. The EFSA Journal, 1041, 1–52.Google Scholar
  19. Eriksson, L., Trygg, J., & Svante, W. (2008). CV-ANOVA for significance testing of PLS and OPLS® models. Journal of Chemometrics, 22, 594–600.CrossRefGoogle Scholar
  20. Gallart Ayala, H., Chéreau, S., Dervilly-Pinel, G., Le Bizec, B. (2015). Potential of mass spectrometry metabolomics for chemical food safety. Bioanalysis Review, 7, 133–146.CrossRefGoogle Scholar
  21. Jacob, C. C., Dervilly-Pinel, G., Biancotto, G., Monteau, F., & Le Bizec, B. (2015). Global urine fingerprinting by LC-ESI(+)-HRMS for better characterization of metabolic pathway disruption upon anabolic practices in bovine. Metabolomics, 11, 184–197.CrossRefGoogle Scholar
  22. JECFA. (2010). Evaluation of data on ractopamine residues in pig tissues. FAO JECFA Monographs 9, meeting 2010.Google Scholar
  23. Jiang, X. F., Zhu, Y. H., & Liu, X. Y. (2014). Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS. Food Additives and Contaminants–Part A Chemistry, Analysis, Control, Exposure and Risk Assessment, 31, 29–38.Google Scholar
  24. Kessner, D., Chambers, M., Burke, R., Agusand, D., & Mallick, P. (2008). ProteoWizard: Open source software for rapid proteomics tools development. Bioinformatics (Oxford, England), 24, 2534–2536.CrossRefGoogle Scholar
  25. Kieken, F., Pinel, G., Antignac, J. P., Monteau, F., Christelle Paris, A., Popot, M. A., et al. (2009). Development of a metabonomic approach based on LC-ESI-HRMS measurements for profiling of metabolic changes induced by recombinant equine growth hormone in horse urine. Analytical and Bioanalytical Chemistry, 394, 2119–2128.CrossRefPubMedGoogle Scholar
  26. Kieken, F., Pinel, G., Antignac, J. P., Paris, A. C., Garcia, P., Popot, M. A., et al. (2011). Generation and processing of urinary and plasmatic metabolomic fingerprints to reveal an illegal administration of recombinant equine growth hormone from LC-HRMS measurements. Metabolomics, 7, 84–93.CrossRefGoogle Scholar
  27. Kouassi Nzoughet, J. J., Dervilly-Pinel, G., Chereau, S., Biancotto, G., Monteau, F., Elliott, C. T., et al. (2015). First insights into serum metabolomics of trenbolone/estradiol implanted bovines; screening model to predict hormone-treated and control animals’ status. Metabolomics, 11, 1184–1196.CrossRefGoogle Scholar
  28. Li, G. L., Fu, Y. H., Han, X. S., Li, X. Y., & Li, C. C. (2016). Metabolomic investigation of porcine muscle and fatty tissue after Clenbuterol treatment using gas chromatography/mass spectrometry. Journal of Chromatography A, 1456, 242–248.CrossRefPubMedGoogle Scholar
  29. Mills, S. E., Kissel, J., Bidwell, C. A., & Smith, D. J. (2003a). Stereoselectivity of porcine badrenergic receptors for ractopamine stereoisomers. Journal of Animal Science, 81, 122–129.CrossRefPubMedGoogle Scholar
  30. Mills, S. E., Spurlock, M. E., & Smith, D. J. (2003b). Beta-adrenergic subtypes that mediate ractopamine stimulation of lipolysis. Journal of Animal Science, 81, 662–668.CrossRefPubMedGoogle Scholar
  31. MOA. (2002). List of banned veterinary drugs and other compounds in food-producing animals. Ministry of Agriculture of China 235th Bulletin.Google Scholar
  32. Nebbia, C., Urbani, A., Carletti, M., Gardini, G., Balbo, A., Bertarelli, D., et al. (2011). Novel strategies for tracing the exposure of meat cattle to illegal growth-promoters. Veterinary Journal, 189, 34–42.CrossRefGoogle Scholar
  33. Parry, T. E. (1957). Paper chromatography of 56 amino compounds using phenol and butanol-acetic acid as solvents with illustrative chromatograms of normal and abnormal urines. Clinica Chimica Acta, 2, 115–125.CrossRefGoogle Scholar
  34. Patti, G. J., Yanes, O., & Siuzdak, G. (2012). Metabolomics: The apogee of the omics trilogy. Nature Reviews Molecular Cell Biology, 13, 263–269.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Pinel, G., Weigel, S., Antignac, J. P., Mooney, M. H., Elliott, C., Nielen, M. W. F., et al. (2010). Targeted and untargeted profiling of biological fluids to screen for anabolic practices in cattle. TrAC Trends in Analytical Chemistry, 29, 1269–1280.CrossRefGoogle Scholar
  36. Ricke, E. A., Smith, D. J., Feil, V. J., Larsen, G. L., & Caton, J. S. (1999). Effects of ractopamine HCl stereoisomers on growth, nitrogen retention, and carcass composition in rats. Journal of Animal Science, 77, 701–707.CrossRefPubMedGoogle Scholar
  37. Riedmaier, I., Becker, C., Pfaffl, M. W., & Meyer, H. H. (2009). The use of omic technologies for biomarker development to trace functions of anabolic agents. Journal of Chromatograohy A, 1216, 8192–8199.CrossRefGoogle Scholar
  38. Smith, C. A., O’Maille, G., Want, E. J., Qin, C., Trauger, S. A., Brandon, T. et al. (2005). METLIN: A metabolite mass spectral database. Therapeutic Drug Monitoring, 27, 747–751.CrossRefPubMedGoogle Scholar
  39. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching and identification. Analytical Chemistry, 78, 779–787.CrossRefPubMedGoogle Scholar
  40. Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211–221.CrossRefPubMedPubMedCentralGoogle Scholar
  41. Tautenhahn, R., Patti, G. J., Kalisiak, E., Miyamoto, T., Schmidt, M., Lo, F. Y., et al. (2011). metaXCMS: Second-order analysis of untargeted metabolomics data. Analytical Chemistry, 83, 696–700.CrossRefPubMedGoogle Scholar
  42. Tautenhahn, R., Patti, G. J., Rinehart, D., & Siuzdak, G. (2012). XCMS online: A web-based platform to process untargeted metabolomic data. Analytical Chemistry, 84, 5035–5039.CrossRefPubMedPubMedCentralGoogle Scholar
  43. van der Kloet, F. M., Bobeldijk, I., Verheij, E. R., & Jellema, R. H. (2009). Analytical error reduction using single point calibration for accurate and precise metabolomic phenotyping. Journal of Proteome Research, 8, 5132–5141.CrossRefPubMedGoogle Scholar
  44. WHO (1993). Ractopamine. Toxicological evaluation of certain veterinary drug residues in food. Food Additives Series No. 31, nos 777 on INCHEM.Google Scholar
  45. Winder, C. L., Dunn, W. B., Schuler, S., Broadhurst, D., Jarvis, R., Stephens, G. M., & Goodacre, R. (2008). Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites. Analytical Chemistry, 80, 2939–2948.CrossRefPubMedGoogle Scholar
  46. Wishart, D. S., Jewison, T., Guo, A. C., Wilson, M., Knox, C., Liu, Y., et al. (2013). HMDB 3.0—The human metabolome database in 2013. Nucleic Acids Research, 41, 801–807.CrossRefGoogle Scholar
  47. Wishart, D. S., Knox, C., Guo, A. C., Eisner, R., Young, N., Gautam, B., et al. (2009). HMDB: A knowledgebase for the human metabolome. Nucleic Acids Research, 37, 603–610.CrossRefGoogle Scholar
  48. Wishart, D. S., Tzur, D., Knox, C., Eisner, R., Guo, A. C., Young, N., et al. (2007). HMDB: The human metabolome database. Nucleic Acids Research, 3, 521–526.CrossRefGoogle Scholar
  49. Wu, Y. P., Bi, Y. F., Bingga, G. L., Li, X. W., Zhanga, S. X., Li, J. C., et al. (2015). Metabolomic analysis of swine urine treated with β2-agonists by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Journal of Chromatography A, 1400, 74–81.CrossRefPubMedGoogle Scholar
  50. Yaeger, M. J., Mullin, K., Ensley, S. M., Ware, W. A., & Slavin, R. E. (2012). Myocardial toxicity in a group of greyhounds administered ractopamine. Veterinary Pathology, 49, 569–573.CrossRefPubMedGoogle Scholar
  51. Zhu, Z. J., Schultz, A. W., Wang, J., Johnson, C. H., Yannone, S. M., Patti, G. J., et al. (2013). Liquid chromatography quadropole-time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database. Nature Protocols, 8, 451–460.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA)LUNAM UniversitéNantesFrance
  2. 2.Chinese Academy of Inspection and Quarantine (CAIQ)BeijingChina

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