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Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids

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Abstract

Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the “invisibility” of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases.

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Acknowledgements

We are grateful to Prof. Dr. Takashi Iida (Nihon University) for the gift of the βUCA synthesized standard. This study was supported, in part, by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01 GM041935 and R35 GM122576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China

    Pingping Zhu, Jian Zhang, Yujie Chen, Shanshan Yin & Ke Lan

  2. Metabolomics Shared Resource, University of Hawaii Cancer Center, Honolulu, HI, 96801, USA

    Mingming Su, Guoxiang Xie & Wei Jia

  3. UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    Kim L. R. Brouwer

  4. State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, China

    Changxiao Liu

  5. Chengdu Health-Balance Medical Technology Co., Ltd., Chengdu, 610000, China

    Ke Lan

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  1. Pingping Zhu
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  2. Jian Zhang
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  3. Yujie Chen
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Corresponding authors

Correspondence to Ke Lan or Wei Jia.

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The human urine and serum samples used in this work were collected from the clinical trial approved by the UNC-CH Biomedical Institutional Review Board and published in ClinicalTrials.gov of U.S. National Library of Medicine (NCT01766960). Informed consent was obtained from the individual participants who provided the urine and serum samples.

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Zhu, P., Zhang, J., Chen, Y. et al. Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids. Anal Bioanal Chem 410, 5287–5300 (2018). https://doi.org/10.1007/s00216-018-1183-7

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