Page 89 - Human Bile Acid Metabolism: a Postprandial Perspective

Short term starvation and bile acid supplementation
Experiment 2. Each subject (n = 10) had two mixed meal tests after an overnight fast on separate study days in a randomly assigned crossover design. In balanced assignment, subjects underwent the control meal test (-gDCA) or received 750 mg of gDCA in capsules concomitantly with the standard meal (+gDCA). We administered a similar dose as used for ursodeoxycholic acid in earlier studies (11, 15). gDCA capsules were prepared for each subject individually under responsibility of the AMC hospital pharmacy.
Mixed Meal Test
For both experiments, subjects were admitted to the unit after an overnight or
40-h fast. The liquid test meal (Nutridrink Compact, Nutricia, Zoetermeer, The
Netherlands) contained 16% protein, 35% fat, and 49% carbohydrates. Subjects
ingested the caloric equivalent of 25% of their estimated daily energy expenditure
calculated using the Harris-Benedict equation. The test meal was ingested at time 5 point 0 (t = 0). In both experiments, blood was sampled from a catheter inserted in
a forearm vein. In experiment 1, blood samples were obtained at 0, 30, 60, 90, 120, 180, and 240 min after meal ingestion and in experiment 2 at -20, -10, 0, 15, 30, 45, 60, 75, 90, 120, 180, and 240 min. Samples were collected into chilled EDTA or heparincoated tubes on ice and immediately centrifuged and subsequently stored at -20°C until analysis. For GLP-1 samples, a dipeptidyl peptidase inhibitor (Ile- Pro-Ile, Sigma-Aldrich, St. Louis, MO) was added to the collection tube at 0.01 mg/mL, and plasma was stored at -80°C.
Laboratory Analysis
Plasma glucose concentrations were analyzed bedside using the glucose oxidation method (EKF Diagnostics, Barleben/Magdeburg, Germany). Insulin was determined on an Immulite 2000 system (Siemens Healthcare Diagnostics, Breda, The Netherlands). Active GLP-1 concentrations were measured by ELISA (EMD Millipore, Billerica, MA). FGF19 was measured at the University of Maastricht using an in-house developed ELISA as published previously (40).
Bile Acid Analysis
Taurine- and glycine-conjugated internal standards [2,2,4,4-2H4]taurocholic acid (tauro-CA), [2,2,4,4-2H4] taurochenodeoxycholic acid (tauro-CDCA), [2,2,4,4- 2H4]glycocholic acid (glyco-CA), and [2,2,4,4-2H4] glycochenodeoxycholic acid (glyco-CDCA) were synthesized as described by Mills et al. (30). Plasma (50 μL)
87