This provides direct evidence that unconjugated bile salts shuttle through peroxisomes to become (re-)conjugated. Reconjugation of deconjugated bile salts is an important process. Over 30% of the total bile salt pool may become deconjugated by intestinal bacteria on a daily basis.4 Our previous study showed that the enzyme catalyzing bile salt conjugation, BAAT, is localized predominantly, if not solely, in peroxisomes.11 This suggests that bile salts need to shuttle through peroxisomes for reconjugation. However, the possible existence of a cytosolic Y-27632 solubility dmso pool of BAAT remains a matter of debate.8-11
Therefore, we developed this novel assay to study trans- and intracellular transport and conjugation of D4CA. The use of D4CA allowed us to specifically follow its (re-)conjugation route, independent from endogenous-produced CA. In the intact liver, bile salt reconjugation is highly efficient, because 97% of Cabozantinib chemical structure infused UDCA is conjugated to taurine or glycine after a single pass through rat livers.5 The in vitro cultured hepatocytes also perform this function with significant efficiency with less than 10% of the D4CA unaccounted for after 24 hours. This part of deuterium-labeled bile salts may still reside in the hepatocytes, be present as (CoA-)intermediate, or is metabolized to other
products. The ratio between the amounts of GCA and TCA formed can be strongly manipulated by the levels of glycine and taurine in the growth medium. A high preference for TCA formation is observed when both amino acids are present in excess. This is in line with the predominant presence of taurine-conjugated bile salts in rats.26 However, in the presence of only glycine GCA is efficiently formed. These data show that the availability Astemizole of glycine and/or taurine strongly determines the final conjugation profile. Glycine-conjugated bile salts are generally more cytotoxic compared to their
taurine equivalents.25 Taurine supplementation in cholestatic diseases may therefore limit liver damage. This is especially relevant in humans who have a predominance of glycine-conjugated bile salts. Cultured rat hepatocytes accumulated high concentrations of D4TCA and D4GCA after exposure to 100 μM D4CA. The cellular volume of a hepatocyte is estimated to be between 4 and 20 pL.19-24 Assuming 20 pL as the volume of a rat hepatocyte, we estimated that the peak intracellular concentrations of D4TCA and D4GCA were ≈200 μM and 400 μM, respectively, whereas D4CA levels were in the range of the concentrations in the medium. The intracellular accumulation of conjugated bile salts is transient, peaks at 3 hours exposure, and then declines. This suggests that export of D4TCA and D4GCA is limiting and cannot keep up with the production of conjugated bile salts. In our model with peroxisomal BAAT, transport of TCA and GCA occurs across the peroxisomal membrane and the plasma membrane (Fig. 7).