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Metabolite quantification: A fluorescence-based method for urine sample normalization prior to 1H-NMR analysis.

James Gerard Wolfsberger1, Emily C Hunt1, Sai Sumedha Bobba2

  • 1Chemistry Department, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, Alabama, 35899, USA.

Metabolomics : Official Journal of the Metabolomic Society
|October 19, 2022
PubMed
Summary

Urine normalization using urobilin concentration improves targeted metabolomics analysis by adjusting for hydration differences. This method narrows metabolite concentration ranges for more accurate results in systems biology research.

Keywords:
1 H-NMR-Urine-Normalization-Urobilin-FluorescenceMetabolomics

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Area of Science:

  • Systems biology
  • Metabolomics
  • Analytical chemistry

Background:

  • Metabolomics offers a snapshot of an organism's metabolic state using techniques like mass spectrometry (MS) and nuclear magnetic resonance (NMR).
  • Accurate metabolite quantification is crucial for understanding biological processes and disease states.

Purpose of the Study:

  • To develop and evaluate a pre-acquisition urine normalization method for targeted metabolomics.
  • To assess the impact of normalization on metabolite concentration variability in rat urine samples.

Main Methods:

  • Urine samples from rats on control and sucrose diets were analyzed.
  • Samples were prepared using two methods: non-normalized and normalized to urobilin concentration.
  • Ten specific metabolites were quantified using an internal standard (DSS) after determining urobilin concentration via fluorescence.

Main Results:

  • Urine normalization improved the pH range for both control and sucrose diet groups.
  • Significant improvement in metabolite concentration range was observed in normalized samples compared to non-normalized samples.
  • All ten quantified metabolites showed significant improvement in range in the sucrose diet group after normalization.

Conclusions:

  • A pre-acquisition urine normalization technique effectively adjusts for hydration state variations.
  • This normalization method leads to a narrower concentration range, enhancing the accuracy of targeted metabolomics analysis.
  • The findings support the utility of urobilin-based normalization for improving the reliability of urine metabolomics studies.