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Evaluation of dilution and normalization strategies to correct for urinary output in HPLC-HRTOFMS metabolomics

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Abstract

Reliable identification of features distinguishing biological groups of interest in urinary metabolite fingerprints requires the control of total metabolite abundance, which may vary significantly as the kidneys adjust the excretion of water and solutes to meet the homeostatic needs of the body. Failure to account for such variation may lead to misclassification and accumulation of missing data in case of less concentrated urine specimens. Here, different pre- and post-acquisition methods of normalization were compared systematically for their ability to recover features from liquid chromatography-mass spectrometry metabolite fingerprints of urine that allow distinction between patients with chronic kidney disease and healthy controls. Methods of normalization that were employed prior to analysis included dilution of urine specimens to either a fixed creatinine concentration or osmolality value. Post-acquisition normalization methods applied to chromatograms of 1:4 diluted urine specimens comprised normalization to creatinine, osmolality, and sum of all integrals. Dilution of urine specimens to a fixed creatinine concentration resulted not only in the least number of missing values, but it was also the only method allowing the unambiguous classification of urine specimens from healthy and diseased individuals. The robustness of classification could be confirmed for two independent patient cohorts of chronic kidney disease patients and yielded a shared set of 49 discriminant metabolite features.

Dilution to a uniform creatinine concentration across urine specimens yields more comparable urinary metabolite fingerprints

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Acknowledgments

This project was funded by the German Federal Ministry of Education and Research (BMBF grant no. 01 ER 0821). We thank the NC, TREAT, and GCKD study participants, the participating nephrologists’ practices and outpatient clinics, as well as the NC, TREAT, and GCKD study personnel and investigators for their enormous commitment. Jerome Rossert was an employee at AMGEN, the sponsor of the TREAT during the conduct of that study.

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Authors and Affiliations

  1. Institute of Functional Genomics, University of Regensburg, Am BioPark 9, 93053, Regensburg, Germany

    Franziska C. Vogl, Sebastian Mehrl, Leonhard Heizinger, Helena U. Zacharias, Lisa Ellmann, Nadine Nürnberger, Wolfram Gronwald, Katja Dettmer & Peter J. Oefner

  2. Department of Epidemiology and Preventive Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93051, Regensburg, Germany

    Inga Schlecht & Michael F. Leitzmann

  3. Thrasos Therapeutics, Inc, 75 Federal Street, Boston, Massachusetts, 02110, USA

    Jerome Rossert

  4. Department of Nephrology and Hypertension, University of Erlangen-Nürnberg, Ulmenweg 18, 91054, Erlangen, Germany

    Kai-Uwe Eckardt

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  1. Franziska C. Vogl
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  2. Sebastian Mehrl
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  3. Leonhard Heizinger
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  4. Inga Schlecht
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  5. Helena U. Zacharias
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  6. Lisa Ellmann
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  7. Nadine Nürnberger
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  8. Wolfram Gronwald
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  9. Michael F. Leitzmann
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  10. Jerome Rossert
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  11. Kai-Uwe Eckardt
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  12. Katja Dettmer
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  13. Peter J. Oefner
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GCKD Study Investigators

Corresponding author

Correspondence to Peter J. Oefner.

Ethics declarations

The studies which provided urine specimens had been approved by the appropriate ethics committees and were performed in accordance with the ethical standards.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

See Electronic Supplementary Material (ESM) for full list of German Chronic Kidney Disease (GCKD) study investigators.

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Vogl, F.C., Mehrl, S., Heizinger, L. et al. Evaluation of dilution and normalization strategies to correct for urinary output in HPLC-HRTOFMS metabolomics. Anal Bioanal Chem 408, 8483–8493 (2016). https://doi.org/10.1007/s00216-016-9974-1

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  • DOI: https://doi.org/10.1007/s00216-016-9974-1

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