Page 90 - Human Bile Acid Metabolism: a Postprandial Perspective

Chapter 5
was diluted with 50 μL internal standard solution (30) and 500 μL acetonitrile was added while mixing. After centrifugation, the sample was dried under N2 and reconstituted in 100 μL methanol:H2O (1:3). Subsequently, 10 μL of this solution was injected onto a UPLC column (Waters Acquity BEH C18; length 10 cm, internal diameter 2.1 mm, particle size 1.7 μm). The BAs were separated using a gradient from 98% 5 mM ammoniumformate (pH 8.1):methanol (3:1 v:v) to 98% acetonitrile:H2O (9:1 v:v) in 5 min at a flow rate of 350 μL/min.
BAs were detected with a Waters Quattro Premier XE tandem mass spectrometer in the negative electrospray ionization mode. For the detection of glycine- and taurine-conjugated BAs, cone voltage was set at 60 V and 90 V, respectively, using 40 eV and 60 eV collision energy, respectively. Overall collision gas pressure was 3 × 10-3 mbar argon, and the source temperature was kept at 120 °C. Glycine-conjugated BAs were detected by multiple reaction monitoring using specific transitions with a mass difference of m/z 74, since all glycine conjugates specifically lose the m/z 74 fragment from the quasimolecular ion after fragmentation (30). Transitions: glyco-CDCA, glyco-DCA, and glyco-ursodeoxycholic acid (UDCA) (448®74); [2H4]glyco-CDCA (452®74); glyco-CA (464®74); [2H4]glyco-CA (468®74). Taurine- conjugated BAs were detected in a similar manner using specific transitions with a mass difference of m/z 80 due to the loss of a part of the taurine moiety. Transitions: tauro-CDCA, tauro-DCA, and tauro-UDCA (498®80); [2H4]tauro- CDCA (502®80); tauro-CA (514®80); [2H4]tauro-CA (518®80). Unconjugated BAs were detected using selected ion recording (cone voltage 70 V, collision energy 10 eV): CDCA and DCA m/z 391, CA m/z 407.
Absolute concentrations of either taurine- or glycine-conjugated CA and CDCA were calculated using calibration curves by relating the peak area to the peak area of the respective 2H4 internal standard. For DCA and UDCA conjugates, the [2H4] CDCA internal standards were used. Calibration curves were labeled linear (r 0.98) from 0.1 to 100 μmol/L.
Energy Expenditure
Energy expenditure (REE) was determined by indirect calorimetry before (at -30 min), 90, and 240 min after the meal using a ventilated hood system (Vmax Encore 29; SensorMedics, Anaheim, CA). Substrate oxidation was calculated as described by Frayn (12). The abbreviated Weir equation was used to calculate 24-h energy expenditure.
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