Abstract
Introduction
Metabolomics is a multi-discipline approach to systems biology that provides a snapshot of the metabolic status of a cell, tissue, or organism. Metabolomics uses mass spectroscopy (MS) and nuclear magnetic resonance (NMR) to analyze biological samples for low molecular weight metabolites.
Objective
Normalize urine sample pre-acquisition to perform a targeted quantitative analysis of selected metabolites in rat urine.
Methods
Urine samples were provided from rats on a control diet (n = 10) and moderate sucrose diet (n = 8) collected in a metabolic cage during an eight hour fast. Urine from each sample was prepared by two different methods. One sample was a non-normalized sample of 1200 µL and the second sample was a variable volume-normalized to the concentration of urobilin in a standard sample of urine. The urobilin concentration in all samples was determined by fluorescence. Ten metabolites for each non-normalized and normalized urine sample were quantified by integration to an internal standard of DSS.
Results
Both groups showed an improvement in pH range going from non-normalized to normalized samples. In the group on the control diet, eight metabolites had significant improvement in range, while the remaining two metabolites had insignificant improvement in range comparing the non-normalized sample to the normalized sample. In the group on the moderate sucrose diet all ten metabolites showed significant improvement in range going from non-normalized to normalized samples.
Conclusions
These findings describe a pre-acquisition method of urine normalization to adjust for differences in hydration state of each organism. This results in a narrower concentration range in a targeted analysis.
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Acknowledgements
The authors would like to thank Dr. John B. Vincent in the Department of Chemistry and Biochemistry at the University of Alabama, Tuscaloosa, for the loan of the metabolic cages used for urine collection. We would also like to thank Genoah Collins, Amelia Clopp, Luis Mercado, and Helen Gibson in Dr. Love-Rutledge’s lab for the rat urine samples used in our experiments.
Funding
This study was financially supported by the Army Education Outreach Program and the Research or Creative Experience for Undergraduates at the University of Alabama in Huntsville.
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JGW wrote the manuscript and prepared figures. SLR, BV contributed to the content of the paper. BV, EH and SB participated in data collection. BV developed NMR method participated in data interpretation, prepared figures. SLR contributed to the design of the study.
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Wolfsberger, J.G., Hunt, E.C., Bobba, S.S. et al. Metabolite quantification: A fluorescence-based method for urine sample normalization prior to 1H-NMR analysis. Metabolomics 18, 80 (2022). https://doi.org/10.1007/s11306-022-01939-y
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DOI: https://doi.org/10.1007/s11306-022-01939-y