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Metabolomics analysis of liver reveals profile disruption in bovines upon steroid treatment

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Abstract

Introduction

The surveillance of illegal anabolic practices in bovine meat production is necessary to guarantee consumers’ health. Screening strategies based on the recognition of indirect biological effects are considered by the community as promising tools to overcome some limitations of classical analytical methods and might therefore concur to ensure safer food for the consumer.

Objectives

The present work aims at characterizing the metabolic profile induced in liver by administration of anabolic steroids, and at identifying potential disturbances in the hepatic metabolism.

Methods

A total of 32 liver samples, 16 from untreated bulls and 16 from bulls treated with an ear implant (Revalor-XS®) containing trenbolone acetate (200 mg) and β-estradiol (40 mg), were analyzed following a LC–MS-based metabolomic analysis combining RP and HILIC chromatographic separations. Different multivariate statistical tools were applied to the datasets to select common metabolites that may be considered as potential markers based on their significant changes in concentrations after administration of sexual steroids.

Results

Eight candidate markers were identified. Moreover, a subset of four markers was also validated by a different laboratory that performed the same analysis using an independent instrumental and elaboration platform, confirming the robustness of the results achieved.

Conclusion

This study was performed mimicking experimental conditions that may be used during a potential misuse practice. It is promising in the objective of setting up an analytical strategy to highlight sexual steroids abuse in livestock animals.

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Abbreviations

PCA:

Principal component analysis

PLS-DA:

Partial least square discriminant analysis

SVM:

Support vector machine

RF:

Random forest

TBA:

Trenbolone acetate

E2:

Estradiol

MS:

Mass spectrometry

HRMS:

High resolution mass spectrometry

HCD:

Higher energy C-trap dissociation

LC:

Liquid chromatography

RP:

Reversed phase

HILIC:

Hydrophilic interaction liquid chromatography

C:

Control

T:

Treated

T/C:

Ratio between treated and control group

ESI:

Electrospray ionization

QC:

Quality control

FWHM:

Full width at half maximum

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Acknowledgements

This work was funded by the Italian Ministry of Health (Project No. IZS VE 2008 RF 1157188, and Project No. IZS VE RC 01/2012 - CUP B28C13000350001). Authors thank Merck Animal Health (NJ, United States) for kindly providing the ear implants Revalor-XS®.

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Authors and Affiliations

  1. Department of Chemistry, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy

    Roberto Stella, Davide Bovo, Roberto Angeletti & Giancarlo Biancotto

  2. Laboratoire d’Étude des Résidus et Contaminants dans les Aliments (LABERCA), ONIRIS, LUNAM Université, Nantes, France

    Gaud Dervilly-Pinel, Anne-Lise Royer & Bruno Le Bizec

  3. Department of Food Safety, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy

    Eleonora Mastrorilli

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  1. Roberto Stella
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  2. Gaud Dervilly-Pinel
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Correspondence to Giancarlo Biancotto.

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Research involving human and animal rights

All institutional and national guidelines for the care and use of laboratory animals were followed. Animals were managed according to Directive 86/609/EEC for the protection of animals used for experimental or other scientific purposes, enforced by the Italian D. Lgs No 116 of January 27, 1992 and Directive 63/2010. The animal study was approved by the Animal Experimentation Ethical Committee of the University of Bologna and was therefore performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

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11306_2017_1220_MOESM1_ESM.eps

Principal component analysis (PCA) performed on metabolic fingerprints to evaluate the quality of the different analytical sequences. Metabolic profiles were acquired applying different chromatographic and ionization conditions: HILIC in positive ionization mode (A), HILIC in negative ionization mode (B), RPLC in positive ionization mode (C), and RPLC in negative ionization mode (D). Quality Control (QC) samples (yellow circles) are clustered together in each of the four plot, indicating the stability and repeatability of the analytical sequence. Frames exhibiting a CV% below 30% in QC samples were used, corresponding to: 777 frames (A), 599 frames (B), 795 frames (C), and 526 frames (D). A 2 components PCA is depicted in each plot. Control samples are represented by blue circles, while treated samples are represented by red circles

Supplementary material 1 (EPS 444 KB)

11306_2017_1220_MOESM2_ESM.eps

Plots reporting the number of principal components against variance value for the PCA performed in order to separate samples (see Fig. 1). For each dataset analyzed using the different instrumental conditions reported in materials and methods section, a chart reports the amount of variance absorbed by each principal component added to the PCA: HILIC in positive ionization mode (A), HILIC in negative ionization mode (B), RPLC in positive ionization mode (C), and RPLC in negative ionization mode (D)

Supplementary material 2 (EPS 592 KB)

Supplementary material 3 (DOCX 13 KB)

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Stella, R., Dervilly-Pinel, G., Bovo, D. et al. Metabolomics analysis of liver reveals profile disruption in bovines upon steroid treatment. Metabolomics 13, 80 (2017). https://doi.org/10.1007/s11306-017-1220-0

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