IMMOBILIZED ARTIFICIAL MEMBRANE CHROMATOGRAPHY - A RAPID AND ACCURATEHPLC METHOD FOR PREDICTING BILE SALT-MEMBRANE INTERACTIONS

To predict bile salt-membrane interactions physiologically, we used an immobilized artificial membrane HPLC column that contains dimyristoyl -phosphatidylcholine molecules covalently linked to silica microsphere s. Using a 90% aqueous (10% acetonitrile) mobile phase, 22 species of bile salts and 4 species of fusidates were eluted. Glycine conjugates displayed higher affinity for the column at pH 5.5, eluting later than their taurine-conjugated congeners, but this order was reversed at pH 6.5 and 7.4 as glycine conjugates became fully ionized. Capacity fact ors decreased logarithmically as functions of increasing temperature, permitting determinations of interaction enthalpies, which ranged from -2.86 to -7.67 kcal/mol. A standard curve was developed from which th e enthalpy for an uncommon bile salt could be inferred from its capaci ty factor at room temperature. Bile salt interaction enthalpies were s ubstantially better correlated than hydrophobic indices by octadecylsi lane-HPLC (D. M. Heuman, J. Lipid Res. 1989. 30: 719-730) with equilib rium binding to small unilamellar vesicles and literature values refle cting bile salt-membrane interactions (e.g., biliary phosphatidylcholi ne secretion), but not with bile salt functions that do not require ph ospholipid (e.g., micellar cholesterol solubility). This new applicati on should prove valuable for evaluating membrane-active physical-chemi cal properties as well as therapeutic potential of novel bile salts, p articularly when they are available in quantities too small for study by conventional techniques.