Home Medicine Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS
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Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS

  • Katharina Habler ORCID logo EMAIL logo , Bernhard Koeppl , Franz Bracher and Michael Vogeser
Published/Copyright: November 22, 2021
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 60 Issue 2

Abstract

Objectives

Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts.

Methods

Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up.

Results

The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) “Guideline on bioanalytical method validation”. To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates.

Conclusions

Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Keywords: bile acids; intrahepatic cholestasis of pregnancy (ICP); liquid chromatography-tandem mass spectrometry (LC-MS/MS); liver disease; profiling; urine
Corresponding author: Dr. rer. nat. Katharina Habler, Institute of Laboratory Medicine, University Hospital, LMU, Marchioninistr. 15, 81377 Munich, Germany, Phone: +49 89 4400 76248, E-mail:

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The local Institutional Review Board deemed the study exempt from review.

References

1. Alnouti, Y. Bile acid sulfation: a pathway of bile acid elimination and detoxification. Toxicol Sci 2009;108:225–46. https://doi.org/10.1093/toxsci/kfn268.Search in Google Scholar

2. Griffiths, WJ, Sjövall, J. Bile acids: analysis in biological fluids and tissues. J Lipid Res 2010;51:23–41. https://doi.org/10.1194/jlr.r001941-jlr200.Search in Google Scholar

3. Gressner, AM, Gressner, OA. Gallensäuren. In: Gressner, AM, Arndt, T, editors. Lexikon der Medizinischen Laboratoriumsdiagnostik. Berlin, Heidelberg: Springer Berlin Heidelberg; 2017.10.1007/978-3-662-49054-9Search in Google Scholar

4. Rassow, J. Biochemie: 50 Tabellen. 2., aktualisierte Aufl. Stuttgart: Thieme; 2008 (Duale Reihe).10.1055/b-002-85529Search in Google Scholar

5. Chiang, JY. Regulation of bile acid synthesis. Front Biosci 1998;3:176–93. https://doi.org/10.2741/a273.Search in Google Scholar

6. Wess, G, Enhsen, A, Kramer, W. Gallensäuren: Wiederentdeckt. Nachr. Chem. Tech. Lab. 1995;43:1047–55. https://doi.org/10.1002/nadc.19950431008.Search in Google Scholar

7. Mayo Clinic Laboratories. Test definition: BAFS: bile acids, fractioned and Tot, S; 2021. Available from: https://www.mayocliniclabs.com/test-catalog/download-setup.php?format=pdf&unit_code=62234.Search in Google Scholar

8. Shima, T, Tada, H, Morimoto, M, Nakagawa, Y, Obata, H, Sasaki, T, et al.. Serum total bile acid level as a sensitive indicator of hepatic histological improvement in chronic hepatitis C patients responding to interferon treatment. J Gastroenterol Hepatol 2000;15:294–9. https://doi.org/10.1046/j.1440-1746.2000.02126.x.Search in Google Scholar

9. Sawkat Anwer, M, Meyer, DJ. Bile acids in the diagnosis, pathology, and therapy of hepatobiliary disease. Vet Clin North Am Small Anim Pract 1995;25:503–17. https://doi.org/10.1016/s0195-5616(95)50039-7.Search in Google Scholar

10. Huang, WM, Tropper, P, Seubert, D, Donnelly, J, Liu, M, Javitt, N. Random urinary bile acids as a diagnostic method for intrahepatic cholestasis of pregnancy (ICP). Am J Obstet Gynecol 2006;195:S121. https://doi.org/10.1016/j.ajog.2006.10.417.Search in Google Scholar

11. Marschall, H-U. Management of intrahepatic cholestasis of pregnancy. Expet Rev Gastroenterol Hepatol 2015;9:1273–9. https://doi.org/10.1586/17474124.2015.1083857.Search in Google Scholar PubMed

12. Tribe, RM, Dann, AT, Kenyon, AP, Seed, P, Shennan, AH, Mallet, A. Longitudinal profiles of 15 serum bile acids in patients with intrahepatic cholestasis of pregnancy. Am J Gastroenterol 2010;105:585–95. https://doi.org/10.1038/ajg.2009.633.Search in Google Scholar

13. Danese, E, Salvagno, GL, Negrini, D, Brocco, G, Montagnana, M, Lippi, G. Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform. PLoS ONE 2017;12:e0179200. https://doi.org/10.1371/journal.pone.0179200.Search in Google Scholar

14. Liu, Y, Rong, Z, Xiang, D, Zhang, C, Liu, D. Detection technologies and metabolic profiling of bile acids: a comprehensive review. Lipids Health Dis 2018;17:121. https://doi.org/10.1186/s12944-018-0774-9.Search in Google Scholar

15. Almé, B, Bremmelgaard, A, Sjövall, J, Thomassen, P. Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry. J Lipid Res 1977;18:339–62. https://doi.org/10.1016/s0022-2275(20)41684-0.Search in Google Scholar

16. Alnouti, Y, Csanaky, IL, Klaassen, CD. Quantitative-profiling of bile acids and their conjugates in mouse liver, bile, plasma, and urine using LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2008;873:209–17. https://doi.org/10.1016/j.jchromb.2008.08.018.Search in Google Scholar

17. Batta, A, Kumar, SG. Gas chromatography of bile acids. J Chromatogr B 1999;723:1–16. https://doi.org/10.1016/s0378-4347(98)00528-3.Search in Google Scholar

18. Amplatz, B, Zöhrer, E, Haas, C, Schäffer, M, Stojakovic, T, Jahnel, J, et al.. Bile acid preparation and comprehensive analysis by high performance liquid chromatography-high-resolution mass spectrometry. Clin Chim Acta 2017;464:85–92. https://doi.org/10.1016/j.cca.2016.11.014.Search in Google Scholar PubMed

19. Luo, L, Aubrecht, J, Li, D, Warner, RL, Johnson, KJ, Kenny, J, et al.. Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans. PLoS ONE 2018;13:1–17. https://doi.org/10.1371/journal.pone.0193824.Search in Google Scholar PubMed PubMed Central

20. Sarafian, MH, Lewis, MR, Pechlivanis, A, Ralphs, S, McPhail, MJW, Patel, VC, et al.. Bile acid profiling and quantification in biofluids using ultra-performance liquid chromatography tandem mass spectrometry. Anal Chem 2015;87:9662–70. https://doi.org/10.1021/acs.analchem.5b01556.Search in Google Scholar PubMed

21. Scherer, M, Gnewuch, C, Schmitz, G, Liebisch, G. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877:3920–5. https://doi.org/10.1016/j.jchromb.2009.09.038.Search in Google Scholar PubMed

22. Burkard, I, Eckardstein, Avon, Rentsch, KM. Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2005;826:147–59. https://doi.org/10.1016/j.jchromb.2005.08.016.Search in Google Scholar

23. Huang, J, Bathena, SPR, Csanaky, IL, Alnouti, Y. Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC-MS/MS. J Pharm Biomed 2011;55:1111–9. https://doi.org/10.1016/j.jpba.2011.03.035.Search in Google Scholar

24. Ducroq, DH, Morton, MS, Shadi, N, Fraser, HL, Strevens, C, Morris, J, et al.. Analysis of serum bile acids by isotope dilution-mass spectrometry to assess the performance of routine total bile acid methods. Ann Clin Biochem 2010;47:535–40. https://doi.org/10.1258/acb.2010.010154.Search in Google Scholar

25. Sangaraju, D, Shi, Y, van Parys, M, Ray, A, Walker, A, Caminiti, R, et al.. Robust and comprehensive targeted metabolomics method for quantification of 50 different primary, secondary, and sulfated bile acids in multiple biological species (human, monkey, rabbit, dog, and rat) and matrices (plasma and urine) using liquid chromatography high resolution mass spectrometry (LC-HRMS) analysis. J Am Soc Mass Spectrom 2021;32:2033–49. https://doi.org/10.1021/jasms.0c00435.Search in Google Scholar

26. Wielders, JPM, Mink, JK. Analysis of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in human urine by high-performance liquid chromatography and fluorometry. J Chromatogr 1984;310:379–85. https://doi.org/10.1016/0378-4347(84)80103-6.Search in Google Scholar

27. European Medicines Agency. Guideline bioanalytical method validation. London, UK: European Medicines Agency; 2011. Available from: http://www.ema.europa.eu/docs/en GB/document library/Scientific guideline/2011/08/WC500109686.pdf.Search in Google Scholar

28. Bonfiglio, R, King, RC, Olah, TV, Merkle, K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:1175–85. https://doi.org/10.1002/(sici)1097-0231(19990630)13:12<1175::aid-rcm639>3.0.co;2-0.10.1002/(SICI)1097-0231(19990630)13:12<1175::AID-RCM639>3.0.CO;2-0Search in Google Scholar

29. Belyakova, LA, Besarab, LN, Roik, NV, Lyashenko, DY, Vlasova, NN, Golovkova, LP, et al.. Designing of the centers for adsorption of bile acids on a silica surface. J Colloid Interface Sci 2006;294:11–20. https://doi.org/10.1016/j.jcis.2005.06.081.Search in Google Scholar

30. Belyakova, LA, Vlasova, NN, Golovkova, LP, Varvarin, AM, Lyashenko, DY, Svezhentsova, AA, et al.. Role of surface nature of functional silicas in adsorption of monocarboxylic and bile acids. J Colloid Interface Sci 2003;258:1–9. https://doi.org/10.1016/s0021-9797(02)00093-0.Search in Google Scholar

31. Tagliacozzi, D. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray. Clin Chem Lab Med 2003;41:1633–41. https://doi.org/10.1515/CCLM.2003.247.Search in Google Scholar

32. Bathena, SPR, Mukherjee, S, Olivera, M, Alnouti, Y. The profile of bile acids and their sulfate metabolites in human urine and serum. J Chromatogr B Analyt Technol Biomed Life Sci 2013;942-943:53–62. https://doi.org/10.1016/j.jchromb.2013.10.019.Search in Google Scholar

33. Perwaiz, S, Tuchweber, B, Mignault, D, Gilat, T, Yousef, IM. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J Lipid Res 2001;42:114–9. https://doi.org/10.1016/s0022-2275(20)32342-7.Search in Google Scholar

34. Simko, V, Michael, S, Kelley, RE. Predictive value of random sample urine bile acids corrected by creatinine in liver disease. Hepatology 1987;7:115–21. https://doi.org/10.1002/hep.1840070123.Search in Google Scholar PubMed

35. Bathena, SPR, Thakare, R, Gautam, N, Mukherjee, S, Olivera, M, Meza, J, et al.. Urinary bile acids as biomarkers for liver diseases I. Stability of the baseline profile in healthy subjects. Toxicol Sci 2015;143:296–307. https://doi.org/10.1093/toxsci/kfu227.Search in Google Scholar PubMed

36. Jahnel, J, Zöhrer, E, Scharnagl, H, Erwa, W, Fauler, G, Stojakovic, T. Reference ranges of serum bile acids in children and adolescents. Clin Chem Lab Med 2015;53:1807–13. https://doi.org/10.1515/cclm-2014-1273.Search in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2021-1111).

Received: 2021-08-12
Accepted: 2021-11-09
Published Online: 2021-11-22
Published in Print: 2022-01-27

© 2021 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2021-1111
Keywords for this article
bile acids; intrahepatic cholestasis of pregnancy (ICP); liquid chromatography-tandem mass spectrometry (LC-MS/MS); liver disease; profiling; urine

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. The clinical value of assessing the inter-method bias: the lesson from prostate specific antigen measurement
  4. Mini Review
  5. Methods to reduce lipemic interference in clinical chemistry tests: a systematic review and recommendations
  6. Opinion Paper
  7. Troponin interference with special regard to macrocomplex formation
  8. Guidelines and Recommendations from Scientific Societies
  9. Use of high-sensitivity cardiac troponins in the emergency department for the early rule-in and rule-out of acute myocardial infarction without persistent ST-segment elevation (NSTEMI) in Italy
  10. Genetics and Molecular Diagnostics
  11. Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening
  12. General Clinical Chemistry and Laboratory Medicine
  13. Comparative study of human growth hormone measurements: impact on clinical interpretation
  14. Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-β peptides 1–42 and 1–40 in CSF
  15. Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid
  16. Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS
  17. High-resolution capillary electrophoresis for the determination of carbamylated albumin
  18. Real-time monitoring of drug laboratory test interactions: a proof of concept
  19. Afamin predicts the prevalence and incidence of nonalcoholic fatty liver disease
  20. Reference Values and Biological Variations
  21. Blood sampling frequency as a proxy for comorbidity indices when identifying patient samples for review of reference intervals
  22. Coagulation parameters in the newborn and infant – the Copenhagen Baby Heart and COMPARE studies
  23. Hematology and Coagulation
  24. Policies and practices in the field of laboratory hematology in Croatia – a current overview and call for improvement
  25. Cardiovascular Diseases
  26. Evaluation of the Atellica TnIH cardiac troponin I assay and assessment of biological equivalence
  27. Infectious Diseases
  28. Inadequate design of mutation detection panels prevents interpretation of variants of concern: results of an external quality assessment for SARS-CoV-2 variant detection
  29. Letters to the Editors
  30. The pronounced decline of anti-SARS-CoV-2 spike trimeric IgG and RBD IgG in baseline seronegative individuals six months after BNT162b2 vaccination is consistent with the need for vaccine boosters
  31. ACE polymorphism is a determinant for COVID-19 mortality in the post-vaccination era
  32. A look at the precision, sensitivity and specificity of SARS-CoV-2 RT-PCR assays through a dedicated external quality assessment round
  33. Value of hypocalcemia and thromboinflammatory biomarkers for prediction of COVID-19 severity during the second wave: were all the waves the same?
  34. Metagenomic next-generation sequencing (mNGS) confirmed a critical case of severe fever with thrombocytopenia syndrome virus (SFTSV)
  35. Biotin interference: evaluation of an updated thyroglobulin electrochemiluminescent immunoassay
  36. Evaluation of the Beckman Coulter Access Procalcitonin Assay: analytical and clinical performance
  37. Analytical performance evaluation of the new sST2 turbidimetric assay implemented in laboratory automation systems
  38. Pre-analytical recommendations and reference values for circulating calprotectin are sample type and assay dependent
  39. Congress Abstracts
  40. Annual Meeting of the Royal Belgian Society of Laboratory Medicine: “Women’s health: from puberty to menopause”
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