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 1H 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 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional 1H–1H TOCSY and 1H–13C 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.

Evidence for a widespread climatic anomaly at around 9.2 ka before present.

The 8.2 ka event was triggered by a meltwater pulse (MWP) into the North Atlantic and resultant reduction of the thermohaline circulation (THC). This event was preceded by a series of at least 14 MWPs; their impact on early Holocene climate has remained almost unknown. A set of high-quality paleoclimate records from across the Northern Hemisphere shows evidence for a widespread and significant climatic anomaly at ∼9.2 ka B.P. This event has climatic anomaly patterns very similar to the 8.2 ka B.P. event, cooling occurred at high latitudes and midlatitudes and drying took place in the northern tropics, and is concurrent with an MWP of considerable volume (∼8100 km3). As the 9.2 ka MWP occurs at a time of enhanced baseline freshwater flow into the North Atlantic, this MWP may have been, despite its relatively small volume, sufficient to weaken THC and to induce the observed climate anomaly pattern.