Page 130 - Human Bile Acid Metabolism: a Postprandial Perspective

Chapter 7
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 60V and 90V respectively, using 40eV and 60eV collision energy respectively. Overall collision gas pressure was 3 x 10ˉ3mbar argon and the source temperature was kept at 120°C. Glycine-conjugated BAs were detected by multiple reaction monitoring (MRM) 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 (29). Transitions: glyco-CDCA, glyco-DCA and glyco-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 70V, collision energy 10eV); 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 labelled linear (r 0.98) from 0.1 to 100μmol/L.
Power analysis and statistics
This study had sufficient power to detect a meal time effect of 180 μmol·min/L on the postprandial area under the curve (AUC) (corresponding to an average change of 1 μmol/L in plasma BA levels) and an effect of group x meal time interaction of 360 μmol·min/L on the AUC (corresponding to an average 2 μmol/L difference between the meal effects of two groups). A 1 μmol/L difference was considered a relevant difference based on in vitro ligand-receptor interaction between BAs and either FXR (14) or TGR5 (30). The study could detect overall group effects on AUC of 1100 μmol·min/L on the AUC (corresponding to a group difference of 6 μmol/L in average plasma BA levels).
Furthermore, the study had sufficient power to detect the following differences in fasting plasma levels: total BAs: 4.7 μmol/L, CA: 0.6 μmol/L, CDCA: 2.7 μmol/L,
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