Christoph Borchers (Presenter)
University of Victoria - Genome BC Proteomics Cent
Bio: Dr. Borchers received his B.S., M.S. and Ph.D. from the University of Konstanz, Germany. After his post-doctoral training and employment as a staff scientist at NIEHS/NIH/RTP in North Carolina, he became the director of the UNC-Duke Proteomics Facility and held a faculty position at the UNC Medical School in Chapel Hill, NC (2001-2006). Since then, Dr. Borchers has been employed at the University of Victoria (UVic), Canada and holds the positions of Professor in the Dept. of Biochemistry & Microbiology and the Rix BC Leadership Chair in Biomedical and Environmental Proteomics. He is also the Director of the UVic – Genome BC Proteomics Centre, which is a member of the Genome Canada funded Genomics Innovation Network. Dr. Borchers is also a Professor at McGill Univ. in the Dept. of Oncology, Montreal, QC where he received the Segal Chair in Mol. Oncology at the Jewish General Hospital.
Authorship: Christoph H. Borchers(1,2), Jun Han(1), Georgia Mitsa(1), and David Hamelin(1)
(1) University of Victoria – Genome BC Proteomics Centre; (2) Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
We applied UPLC-MS/MS techniques using class-specific multiple-reaction monitoring on a triple-quadrupole mass spectrometer and using high-energy collision-induced dissociation and neutral loss- and precursor ion-initiated MS/MS/MS on an LTQ-Orbitrap mass spectrometer to comprehensively profile conjugated and unconjugated bile acids in various human and mouse samples. UPLC fractionation and enzymatic de-conjugation was used to assist in the identification. Chemical derivatization –UPLC-MS/MS was performed to detect and identify of structural isomers of mono-/di-/tri-OH bile acids. As a result, >80 bile acids with potentially uncharacterized structures have been detected and confirmed, in addition to 69 known bile acids.
Bile acids (BAs) are steroidal end products that are synthesized from cholesterol in hepatocytes and play key roles in human physiology, such as emulsification of dietary fat and fat-soluble vitamins, regulation of their own synthesis and energy metabolism homeostasis. Lipophilic BAs at abnormal physiological concentrations also have a wide range of cytotoxicity that is associated with various human pathologies. Therefore, comprehensive detection, identification, and quantitation of BAs and their related endogenous metabolites has the potential to be used for diagnosis, prognosis, and management of liver diseases and many other disorders. This work focuses on the comprehensive profiling of >150 bile acids of known and unknown structures in different human and mouse biological samples, using UPLC-MS/MS and related techniques.
A dual-gradient reversed-phase UPLC/multiple reaction monitoring (MRM)-MS method was optimized to separate 32 unconjugated, 8 glycine-conjugated, 12 taurine-conjugated, 13 sulfated, and 4 glucuronidated BA standards. The Q1 to Q3 ion transitions for each of the isomeric BA groups were then used to construct class-specific MRM lists for the untargeted analysis of tetrahydroxylated (TH) BAs and the conjugated BAs of unknown structures in human blood, urine and feces as well as in mouse blood and liver. Chemical derivatization with 3-nitrophenylhydrazine was used for the UPLC/MRM-MS detection of the unconjugated BAs with both known and unknown structures. Confirmation and identification of the detected putative BAs was performed using combined UPLC fractionation, enzymatic deconjugation, and UPLC-high-resolution MS/MS.
Separation of the 69 known BA standards was achieved in a 25-min gradient by UPLC on a 15-cm reversed-phase C18 column (2.1 x 150 mm, 1.7 μm) at 45 oC and with dual (pH and organic solvent)-gradient elution by UPLC/MRM-MS on a triple-quadrupole mass spectrometer. The use of class-specific UPLC/MRM-MS resulted in the successful detection of >50 unconjugated THBAs and amino acid (glycine and taurine)-conjugated BAs with unknown structures in the mouse samples, and at least 30 such species in the human samples. The detection of these putative BAs was confirmed by UPLC-high-resolution MS and MS/MS using high-energy collision-induced dissociation as well as neutral loss- and precursor ion-triggered MS3 on an LTQ-Orbitrap mass spectrometer. The use of chemical derivatization prior to UPLC-MS/MS enabled the MRM detection of at least 15 putative unconjugated mono-, di-, and tri-OH BAs and 23-carbon-atom nor-BAs in the tested samples.
Human blood, urine, and fecal samples showed significantly different BA profiles. For example, the BA components in human urine were dominated by esterified metabolites (i.e., sulfates and glucuronides), and many of the detected sulfates and glucuronides were not among the known BA standards used in this study. The major BAs detected in human fecal samples were secondary BAs (deoxycholic and lithocholic acid) and their hydrophobic isomers together with many oxo-, keto- and dehydorxylated BAs, resulting from co-metabolism regulated by gut microbiota.
To identify the putative BA glucuronides, UPLC fractionation was performed in combination with enzymatic deconjugation using β-glucuronidase, followed by re-analysis of the deconjugated fractions by UPLC/MRM-MS. In this way, ursodeoxycholic acid-3-glucuronide (UDCA-3-G) and UDCA-24-G were successfully identified. The authentic compounds of various BA sulfates are currently being synthesized in order to structural confirmation of the detected BA sulfates.
The BA compositions of human and mouse samples were unexpectedly complex and many BAs were quantified for the first time in this work. Based on these results, systematic approaches were proposed for comprehensive and reliable analysis of BAs in various human and mouse samples, including the development and validation of a “46 Bile Acids MRM Assay Kit”.
References & Acknowledgements:
This work was supported by funding to The Metabolomics Innovation Centre (TMIC) from Genome Canada, Genome BC, and Genome Alberta for operations (205MET and 7203) and technology development (215MET and MC3T).
|Grants||yes||Genome Canada, Genome BC, Genome Alberta|
|Salary||yes||University of Victoria|
|Board Member||yes||Editorial Boards for scientific journals and scientific organizations (e.g., HUPO)|
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||