Page 52 - Human Bile Acid Metabolism: a Postprandial Perspective

Chapter 3
Glucose and insulin concentrations
We measured glucose concentrations in duplicate with a glucose oxidase method using the Biosen C-line plus glucose analyser (EKF Diagnostics, Barleben/ Magendeburg, Germany) and insulin concentrations in duplicate with a porcine Insulin ELISA (version 4.0, Mercodia, Uppsala, Sweden) according to the manufacturer protocol. Plates were read with a spectrophotometer (Varioskan Flash version 2.4.3, Thermo Scientific) running matching SkanIt software. The calibration curve was a cubic polynomial extrapolated to concentrations of 0.01-2.0 ng/L. Samples outside this range were set to ≤ 0.01 or ≥ 2.0 ng/L, respectively. Insulin concentrations were not measured in renal vein samples.
BA concentrations
We measured BA concentrations by liquid chromatographytandem mass spectrometry (LC/MS/MS, Supplemental Methods). Pigs have an abundant BA profile [17,18]. Here, we focused mainly on BAs that are prevalent in humans to enable translation: ursodeoxycholic acid (UDCA), cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and hyodeoxycholic acid (HDCA, non-human), and their glycine- (g) and taurine- (t) conjugated forms. In the humans, 15 BAs were measured: UDCA, CA, CDCA, DCA and LCA and their g- and t- conjugated forms. For sample preparation, after homogenizing and spinning, 25 mL plasma was aliquoted into a clean tube for BA analysis. For every 10 samples prepared, one quality control standard plasma was included. To each sample, we added 250 mL internal standard and vortexed for 60 s. Samples were centrifuged at 15,800 ×g and the supernatant poured into a clean glass tube.
The fluid was evaporated under nitrogen at 40 °C. Before measuring samples were reconstituted in 100 mL 50% methanol in water, vortexed for 60 s and centrifuged for 3 min at 1800 ×g. We transferred the supernatant into a 0.2 mm spin-filter and centrifuged at 2000 ×g for 10 min. After filtering, the samples were transferred into LC/MS vials and analysed (10 mL injection volume). The lower limit of quantification (LOQ) was 0.05 mM for all BA forms.
In pigs, all measured concentrations of CA, tCA, gCA, DCA, gDCA and tDCA were below the LOQ. Unconjugated UDCA concentration was never calculated due to interfering peaks in the chromatogram. Therefore, we excluded the
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