Spread Monomolecular Films of Monohydroxy Bile Acids and Their Salts:  Influence of Hydroxyl Position, Bulk pH, and Association with Phosphatidylcholine

Undissociated dihydroxy bile acids, alone or with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), lie with their long axes parallel to aqueous−lipid interfaces [Fahey, D. A., Carey, M. C., and Donovan, J. M. (1995) Biochemistry 34, 10886−10897]. To test the generality of this orientation, we used an automated Langmuir−Pockels surface balance to examine pressure−molecular area isotherms and dipole moments of insoluble monohydroxy bile acids and their salts, which are sparingly soluble because of their presumed high Krafft points. We studied lithocholic acid (LCA) (the natural 3α-OH isomer), glycolithocholic acid (GLCA) (its glycine conjugate), and the semisynthetic isomers, 7α-OH- and 12α-OH-cholanoic acids with and without POPC, at pH values ranging from 2 to 12. Monolayer collapse pressures increased sigmoidally with ionization, giving apparent pK values of 7.0−8.5 and implying a stronger affinity of the bile salt anions for the interface. At monolayer collapse, the molecular area of LCA was ∼85 Å2 independent of pH, consistent with the steroid nucleus lying flat. In contrast, the interfacial area of 7-OH-cholanoic acid decreased from ∼80 Å2 at pH 2 to ∼40 Å2 above pH 9, consistent with a more vertical orientation and approximating 12-OH-cholanoic acid, which exhibited a molecular area of ∼45 Å2 at all pH values. All monohydroxy bile acids condensed POPC monolayers more effectively at low than at high (ionized) pH. We conclude that the 3-OH group is crucial for anchoring bile acids and their salts to the aqueous interface, with all monohydroxy species condensing phospholipid membranes regardless of ionization state.