EFFECTS OF SIDE-CHAIN LENGTH ON IONIZATION BEHAVIOR AND TRANSBILAYER TRANSPORT OF UNCONJUGATED DIHYDROXY BILE-ACIDS - A COMPARISON OF NOR-CHENODEOXYCHOLIC ACID AND CHENODEOXYCHOLIC ACID

C-13-NMR spectroscopy was used to examine the effect of side chain length on the ionization properties and transmembrane transport rate of 3 alpha,7 alpha-dihydroxy bile acids. When solubilized in taurocholate micelles, [23-C-13]nor-chenodeoxycholic acid (nor-CDCA) had a pK(a) of 6.1, similar to that of CDCA (pK(a) 6.2), its C-24 homologue. In unilamellar phosphatidylcholine vesicles, the pK(a) of nor-CDCA was 7.0, whereas that of CDCA was 6.6. Lineshape analysis indicated that the rate of ionization of nor-CDCA as a micellar solute or as a vesicle component was very slow (0.4 x 10(5) sec(-1)) compared to that of acetic acid in water (8.7 x 10(5) sec(-1)). Lineshape analysis of spectra of the protonated form of nor-CDCA at acidic bulk pH indicated that the transbilayer transport rate of nor-CDCA (580 sec(-1)) was six times faster than that of CDCA (100 sec(-1)). It is proposed that the shorter side chain of the nor-CDCA molecule causes it to reside more deeply inside the Vesicle bilayer than CDCA, explaining its weaker ionization and more rapid flip-flop rate. These in vitro experiments imply that, in vivo, a given C-23 nor-dihydroxy bile acid will ionize less readily when present in membranes, and it will also Aip-flop faster than its C-24 homologue.