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Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples

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Abstract

Bile acids (BAs) are a complex class of metabolites that have been described as specific biomarkers of gut microbiota activity. The development of analytical methods allowing the quantification of an ample spectrum of BAs in different biological matrices is needed to enable a wider implementation of BAs as complementary measures in studies investigating the functional role of the gut microbiota. This work presents results from the validation of a targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 28 BAs and six sulfated BAs, covering primary, secondary, and conjugated BAs. The analysis of 73 urine and 20 feces samples was used to test the applicability of the method. Concentrations of BAs in human urine and murine feces were reported, ranging from 0.5 to 50 nmol/g creatinine and from 0.012 to 332 nmol/g, respectively. Seventy-nine percent of BAs present in human urine samples corresponded to secondary conjugated BAs, while 69% of BAs present in murine feces corresponded to primary conjugated BAs. Glycocholic acid sulfate (GCA-S) was the most abundant BA in human urine samples, while taurolithocholic acid was the lowest concentrated compound detected. In murine feces, the most abundant BAs were α-murocholic, deoxycholic, dehydrocholic, and β-murocholic acids, while GCA-S was the lowest concentrated BA. The presented method is a non-invasive approach for the simultaneous assessment of BAs and sulfated BAs in urine and feces samples, and the results will serve as a knowledge base for future translational studies focusing on the role of the microbiota in health.

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Acknowledgements

The authors would like to thank all study participants, as well as the nurses and medical staff involved, for their effort and commitment.

Funding

This work was funded by the European Union’s Horizon 2020 Research and Innovation Programme through the Nutrishield project (https://nutrishield-project.eu/) [Grant Agreement No 818110] and the Carlos III Health Institute (Ministry of Science and Innovation, Spain) and co-funded by the European Union [grant numbers CD19/00176, and CPII21/00003], grant Despega IATA from IATA-CSIC, the Spanish Ministry of Science and Innovation [grant number PID2020-119536RB-I00], and MCIN/AEI/https://doi.org/10.13039/501100011033 and by ERDF A way of making Europe, by the “European Union” [grant number PID2021-125573OB-I00].

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Authors and Affiliations

  1. Neonatal Research Unit, Health Research Institute Hospital La Fe, Avda Fernando Abril Martorell 106, 46026, Valencia, Spain

    Victoria Ramos-Garcia, Isabel Ten-Doménech, Máximo Vento & Julia Kuligowski

  2. Division of Neonatology, University & Polytechnic Hospital La Fe, Avda Fernando Abril Martorell 106, 46026, Valencia, Spain

    Máximo Vento

  3. Institute of Agrochemistry and Food Technology, Spanish National Research Council (IATA-CSIC), Valencia, Spain

    Clara Bullich-Vilarrubias, Marina Romaní-Pérez & Yolanda Sanz

  4. Autoimmune Pathogenesis Unit, IRCCS San Raffaele Scientific Institute, 20132, Milan, Italy

    Angelica Nobili & Marika Falcone

  5. Maternal and Child Health Area, IRCCS San Raffaele Scientific Institute, 20132, Milan, Italy

    Marina Di Stefano

  6. Health and Biomedicine, Leitat Technological Center, Carrer de la Innovació, 2, 08225, Terrassa, Spain

    Guillermo Quintás

Authors
  1. Victoria Ramos-Garcia
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Contributions

Conceptualization: Julia Kuligowski and Guillermo Quintás. Methodology: Victoria Ramos-Garcia. Formal analysis: Victoria Ramos-Garcia, Guillermo Quintás. Data curation: Victoria Ramos-Garcia, Guillermo Quintás. Writing—original draft preparation: Victoria Ramos-Garcia, Clara Bullich-Vilarrubias, Marina Romaní-Pérez, Yolanda Sanz, Angelica Nobili, Marika Falcone, Marina Di Stefano. Writing—review and editing: all authors. Supervision: Julia Kuligowski, Isabel Ten-Doménech, and Máximo Vento. Project administration: Julia Kuligowski. Funding acquisition: Julia Kuligowski. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Julia Kuligowski.

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Ethics approval

During the enrolment phase, pediatricians informed the parents about the purpose of this study, the lack of reported risks related to the collection of samples, the effort required to take part in this study, and their right to deny or withdraw their consent at any time. Parents accepted to participate in the study and signed an informed consent form. The study was approved by the Institutional Ethical Committee of the IRCCS San Raffaele Scientific Institute (Protocol: NUTRI-T1D, 2019). The experimental procedure using animals was in accordance with European Union 2010/63/UE and Spanish RD53/2013 guidelines and approved by the ethics committee of the University of Valencia (Animal Production Section, SCSIE, University of Valencia, Spain) and authorized by Dirección General de Agricultura, Ganadería y Pesca (Generalitat Valenciana) (approval ID 2021/VSC/PEA/0273).

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Ramos-Garcia, V., Ten-Doménech, I., Vento, M. et al. Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples. Anal Bioanal Chem 415, 4961–4971 (2023). https://doi.org/10.1007/s00216-023-04802-8

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