Page 51 - Human Bile Acid Metabolism: a Postprandial Perspective

Sample preparation
After withdrawal, we immediately placed the blood samples on ice. For the PAH concentration measurements, we pipetted 250 mL of blood into a tube containing
25 mL of trichloroacetic acid (TCA), thoroughly vortexed, and subsequently,
together with the remaining blood samples, centrifuged at 8000 g for 5 min at 4 °C.
After spinning, we transferred the plasma samples into clean tubes and snap
froze them in liquid nitrogen and stored them at 80 °C until further analysis. 3 For measurements, samples were defrosted and thoroughly homogenized using
a multivortex and subsequently centrifuged for 3 min at 1780 or 1800 g and aliquoted for BA, insulin and glucose concentration measurements.
PAH concentrations and flow calculations
The PAH concentrations of the TCA plasma samples were compared to PAH standards and read out using a microplate spectrophotometer (Spectramax; Molecular Devices, Sunnyvale, CA) and SoftmaxPro software (Molecular Devices) [16]. We calculated plasma flow through the organs using the dilution of PAH over the organ compartment [14,15]. Blood sampling (Fig. 1) started when PAH concentrations reached steady state (60 min). So PAHIN PAHOUT. We calculated the plasma flow through the hindquarter muscle (HQ), the intestine (portal drained viscera, PDV) and the splanchnic compartment (PDV and liver, SPL) with the formula:
For HQ the PAH infusion site is the catheter in the abdominal aorta. For PDV and SPL the infusion site is the catheter into the splenic vein. [PAH]pre is the PAH concentration in the main bloodstream, sampled through line A. [PAH]post is the PAH concentration in the efferent vein of the organ: line P for PDV, line H for SPL and line V for HQ. Plasma flow through the kidneys (K) can be calculated with the formula:
Note that for K the extracted PAH is the infused [PAH] from both infusion sites. We interpolated missing data points and used the mean plasma flow of all pigs for further flux calculations.
Transhepatic bile acid kinetics
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