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Standardization of factors that influence human urine metabolomics

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Abstract

In nutritional metabolomics a large inter- and intra-subject variability exists, and thus, it becomes important to limit the variance introduced by external factors. In a composite controlled study with full provision of all food for the standardized intervention, human urinary metabolite profiles were investigated for different factors, such as handling of urine collections, diet standardization, diet culture, cohabitation and gender. In study A, 8 healthy subjects (4 men; 4 women) collected 24-h urine, splitting each void into two specimens stored either at 4°C or at room temperature. In study B, 16 healthy subjects (7 men; 9 women) collected 24-h urine for three days while being on a standardized diet. Samples were analyzed by 1H NMR and chemometrics. The NMR profiles indicated the presence of metabolites presumably originating from bacterial contamination in 3 out of 16 sample collections stored at room temperature. On the contrary, no changes in the NMR profiles due to contamination occurred in the 24-h urine samples stored at 4°C. The study also showed a trend towards a reduced inter- and intra-individual variation during 3 days of diet standardization. In study A, the urine metabolome showed a clear effect of diet culture and cohabitation, but these effects significantly attenuated after diet standardization (study B). Besides, gender-specific differences were found in both studies. Our results emphasize that best practice for any metabolomic study is a standardized, chilled sample collection procedure, and recommend that diet standardization is performed prior to dietary interventions in order to reduce intra- and inter-subject variability.

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Acknowledgments

The Danish Ministry of Food, Agriculture and Fisheries is acknowledged for sponsoring the project “Urinary biomarkers for eating patterns using NMR spectroscopy and Chemometrics” (3304-FVFP-060706-01) under which the present research has been developed and the Villum Kann Rasmussen project (Metabonomic Cancer Diagnostic) for continuing the funding for FS. LGR was supported by a research grant from the EU project DiOGenes and a research grant from the project BEST at the University of Copenhagen. LGR conducted the trial and FS conducted the 1H NMR analyses. FS and SBE conducted the chemometric analyses. SBE, FS and LGR were responsible for the interpretation of the results. LGR and FS authored the manuscript with co-authorship from TML, LOD, AA and SBE.

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Authors and Affiliations

  1. Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 1958, Frederiksberg C, Denmark

    Lone G. Rasmussen, Thomas M. Larsen, Lars O. Dragsted & Arne Astrup

  2. Quality and Technology, Department of Food Science Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark

    Francesco Savorani & Søren B. Engelsen

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  1. Lone G. Rasmussen
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  2. Francesco Savorani
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  6. Søren B. Engelsen
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Correspondence to Lone G. Rasmussen.

Additional information

Lone G. Rasmussen, Francesco Savorani contributed equally to this work.

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11306_2010_234_MOESM1_ESM.ppt

“Supplementary data”—online version Supplementary Figure 1. 500 MHz 1H NMR spectrum of human urine split in three regions: high-field, 0.8–2.95 ppm (A); mid-field, 2.95–4.1 ppm (B); low-field, 6.3–8.95 ppm (C). Vertical scale in A and C are respectively magnified 10 and 100 times, with respect to B. The spectrum is a mean of the samples from both study A and B. Assignments of the most significant peaks are numbered and the corresponding metabolites are listed in Supplementary Table 2. Supplementary Figure 2. Cohabitation effect for study B. PCA scores plot in which samples are colored according to couples of subjects living together (cohabitation). No significant clustering effect can be found confirming that standardization of the diet was effective on equalizing urinary metabolomes. Supplementary Figure 3. iECVA spectrum of the 1H NMR urinary data set for study B highlighting the marker alanine, responsible of diet culture differentiation (*): its spectral features are showed in the inset. Supplementary Figure 4. Gender discrimination for study A. A: PCA scores plot showing that the classification according to gender is achieved in PC#1 vs. PC#3. B: iECVA spectrum highlighting the markers responsible of gender differentiation (*): creatinine, citrate and alanine spectral features are illustrated in the above insets. Supplementary Figure 5. Gender discrimination for study B. PCA scores plot of the whole 1H NMR dataset in which samples are marked according to gender. No complete discrimination is achieved but a strong tendency towards gender separation can be found along both PC#1 and PC#5. (PPT 1336 kb)

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Rasmussen, L.G., Savorani, F., Larsen, T.M. et al. Standardization of factors that influence human urine metabolomics. Metabolomics 7, 71–83 (2011). https://doi.org/10.1007/s11306-010-0234-7

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