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Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS
Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Bile/metabolism, Bile Acids and Salts/chemistry, Chromatography, High Pressure Liquid/methods, Chromatography, Liquid/methods, Fatty Liver/metabolism, Humans, Liver/metabolism, Magnetic Resonance Spectroscopy/methods, Mass Spectrometry/methods, Metabolome, Models, Chemical, Reproducibility of Results
1742-2051
180-190
Duarte, Iola F
aa485146-f35f-46df-acc8-f67a57e4f6fc
Legido-Quigley, Cristina
8bff865c-d50d-4830-934f-64daa5cafb90
Parker, David A
fe049bae-f809-4564-b614-c13de07e3a0c
Swann, Jonathan R
7c11a66b-f4b8-4dbf-aa17-ad8b0561b85c
Spraul, Manfred
695b39fc-18f9-4388-8abc-d3948de3e4d3
Braumann, Ulrich
52c35b93-56ab-4cdf-a3ef-61e826cb1f14
Gil, Ana M
7c029765-ce52-4438-a591-1e55c85312bc
Holmes, Elaine
d3b92a6b-1c3f-4758-b653-ba35afd3f57d
Nicholson, Jeremy K
72991774-7e08-4592-ae57-e7dcc2ec158e
Murphy, Gerard M
5a1d2cfa-3e5a-4b83-aa2e-6093fe7ef630
Vilca-Melendez, Hector
63497806-dd34-4db3-bded-d59e10096e8b
Heaton, Nigel
d35bd960-5c3a-4035-9794-7b493ac89014
Lindon, John C
9c93c665-3190-426c-a2d5-1805d8a7900c
Duarte, Iola F
aa485146-f35f-46df-acc8-f67a57e4f6fc
Legido-Quigley, Cristina
8bff865c-d50d-4830-934f-64daa5cafb90
Parker, David A
fe049bae-f809-4564-b614-c13de07e3a0c
Swann, Jonathan R
7c11a66b-f4b8-4dbf-aa17-ad8b0561b85c
Spraul, Manfred
695b39fc-18f9-4388-8abc-d3948de3e4d3
Braumann, Ulrich
52c35b93-56ab-4cdf-a3ef-61e826cb1f14
Gil, Ana M
7c029765-ce52-4438-a591-1e55c85312bc
Holmes, Elaine
d3b92a6b-1c3f-4758-b653-ba35afd3f57d
Nicholson, Jeremy K
72991774-7e08-4592-ae57-e7dcc2ec158e
Murphy, Gerard M
5a1d2cfa-3e5a-4b83-aa2e-6093fe7ef630
Vilca-Melendez, Hector
63497806-dd34-4db3-bded-d59e10096e8b
Heaton, Nigel
d35bd960-5c3a-4035-9794-7b493ac89014
Lindon, John C
9c93c665-3190-426c-a2d5-1805d8a7900c

Duarte, Iola F, Legido-Quigley, Cristina, Parker, David A, Swann, Jonathan R, Spraul, Manfred, Braumann, Ulrich, Gil, Ana M, Holmes, Elaine, Nicholson, Jeremy K, Murphy, Gerard M, Vilca-Melendez, Hector, Heaton, Nigel and Lindon, John C (2009) Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. Molecular BioSystems, 5 (2), 180-190. (doi:10.1039/b814426e).

Record type: Article

Abstract

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

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More information

Accepted/In Press date: 20 November 2008
e-pub ahead of print date: 15 December 2008
Published date: February 2009
Keywords: Bile/metabolism, Bile Acids and Salts/chemistry, Chromatography, High Pressure Liquid/methods, Chromatography, Liquid/methods, Fatty Liver/metabolism, Humans, Liver/metabolism, Magnetic Resonance Spectroscopy/methods, Mass Spectrometry/methods, Metabolome, Models, Chemical, Reproducibility of Results

Identifiers

Local EPrints ID: 439398
URI: http://eprints.soton.ac.uk/id/eprint/439398
ISSN: 1742-2051
PURE UUID: 6bc0a413-56a5-425d-bd73-4c13f5c5a4b2
ORCID for Jonathan R Swann: ORCID iD orcid.org/0000-0002-6485-4529

Catalogue record

Date deposited: 21 Apr 2020 16:52
Last modified: 26 Nov 2021 03:21

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Contributors

Author: Iola F Duarte
Author: Cristina Legido-Quigley
Author: David A Parker
Author: Manfred Spraul
Author: Ulrich Braumann
Author: Ana M Gil
Author: Elaine Holmes
Author: Jeremy K Nicholson
Author: Gerard M Murphy
Author: Hector Vilca-Melendez
Author: Nigel Heaton
Author: John C Lindon

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