4). Excess of either glycine or taurine in the culture medium leads to a concomitant D4GCA and D4TCA production, respectively, both extracellularly (Fig. 4A) and intracellularly (Fig. 4B). When both glycine and taurine were present in excess in the medium, D4CA was predominantly converted to D4TCA (70 μM, compared to only 2 μM D4GCA). Peak accumulation of D4TCA (200 μM) and D4GCA (400 μM) in hepatocytes was observed after 3 hours exposure to D4CA (Fig. 2). Hepatocytes exposed to these conditions were analyzed by

digitonin permeabilization assays to determine whether D4-labelled bile salts accumulate in membrane-enclosed intracellular compartments. Low concentrations of digitonin (30 μg/mL) disrupt the plasma membrane and cytosolic components are effectively released from the cellular fraction

(Fig. INCB024360 5A; glyceraldehyde 3-phosphate dehydrogenase [GAPDH] is shown as a cytosolic marker protein, quantification in Fig. 5B). D4CA and D4GCA are fully released from hepatocytes at this concentration (Fig. 5B, shown only for D4CA). The peroxisomal membrane is more resistant to digitonin permeabilization and is only fully permeabilized at 500 μg/mL. Partial release of the peroxisomal marker proteins catalase and BAAT is observed Doxorubicin at digitonin concentrations of 30 and 150 μg/mL (Fig. 5A, quantification in Fig. 5B). The digitonin-extractability of D4TCA lies between the profile for GAPDH/D4CA and catalase (Fig. 5B), suggesting that D4TCA accumulates, at least partly, in membrane-enclosed

organelles with peroxisomal characteristics. To obtain further evidence for the accumulation of D4TCA in peroxisomes, we purified these organelles Protirelin from a PNS fraction of D4CA-exposed rat hepatocytes (Fig. 6). After Nycodenz density gradient centrifugation of the PNS, all 20 gradient fractions were analyzed for the presence of D4TCA, D4CA and markers for various cellular compartments. A PMP70/BAAT-enriched peak was detected at high density fractions 3-5, separated from mitochondria (Cyt C; fractions 10-11) and cytosol (GAPDH; fractions 15-20) (Fig. 6A). The highest concentrations of D4TCA were detected at the top of the gradient (Fig. 6B). In addition, minor but significant amounts of D4TCA were detected in fractions 3-5, revealing a similar concentration profile as the peroxisomal marker proteins (Fig. 6C). In contrast, D4CA and D4GCA were not detected in the peroxisome-enriched fractions. In this study we established a novel assay that allows the study of transcellular and intracellular transport and conjugation of bile salts by rat hepatocytes in vitro. Primary rat hepatocytes effectively convert exogenously added D4CA to its D4TCA and D4GCA.