The human apical sodium-dependent bile acid transporter (hASBT) is a potential target for drug delivery, but an understanding of hASBT substrate requirements is lacking. The objective of this study was to characterize hASBT interaction with its native substrates, bile acids, and to evaluate C-24 conjugation and steroidal hydroxylation on transport affinity and inhibition potency.

Transport and inhibition kinetics of 15 bile acids were evaluated (cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate, and lithocholate, including their glycine and taurine conjugates) with an hASBT–Madin-Darby canine kidney (MDCK) monolayer assay. Samples were analyzed via liquid chromatography–mass spectrometry (LC-MS) or chromatography–mass spectrometry–mass spectrometry (LC-MS-MS).

C-24 conjugation improved the inhibitory potency of all native bile acids. There was an inverse association between number of steroidal hydroxyl groups and inhibitory potency. Glycolithocholate and taurolithocholate were the most potent inhibitors. Results from transport studies followed trends from inhibition studies. Conjugated dihydroxy and monohydroxy bile acids exhibited the highest hASBT-mediated transport (i.e., lower K _t and higher J _max). Across the 15 bile acids, K _t generally followed K _i. Additionally, J _max correlated with K _i, where greater inhibition potency was associated with higher transport capacity.

C-24 conjugation and steroidal hydroxylation pattern modulated native bile acid interaction with hASBT, with C-24 effect dominating steroidal hydroxylation effect. Results indicate that bile acid binding to hASBT may be the rate-limiting step in the apical transport of bile acids.