resumo
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 H-1-H-1 TOCSY and H-1-C-13 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.
palavras-chave
NUCLEAR-MAGNETIC-RESONANCE; LIVER-TRANSPLANTATION; STEP ANALYSIS; ACIDS; DISEASE; H-1; QUANTIFICATION; SECRETION; PROTON; SALT
categoria
Biochemistry & Molecular Biology
autores
Duarte, IF; Legido-Quigley, C; Parker, DA; Swann, JR; Spraul, M; Braumann, U; Gil, AM; Holmes, E; Nicholson, JK; Murphy, GM; Vilca-Melendez, H; Heatond, N; Lindon, JC
nossos autores
agradecimentos
We thank the Roche Organ Transplantation Research Foundation (ROTRF) for financial support to CLQ and DAP and the Foundation for Science and Technology, Portugal, for funding support through the grant SFRH/BPD/30337/2006 within the III Community framework (to IFD). Funding from the British Council/CRUP-Portugal (Integrated Action B54 06) is also gratefully acknowledged.