Dihydroxy Salts Research Articles

Bile salt- and lysophosphatidylcholine-induced membrane damage in human erythrocytes.

The interaction of bile salts and lysophosphatidylcholine (LPC) with membranes has implications both in understanding the aetiology of a number of gastrointestinal disorders, including gastritis, gastric ulcers and colonic cancer, and in enhancing drug absorption by various epithelia. The membrane toxicity of nine bile salts (the sodium (S) salts of chenodeoxycholate (CDC), deoxycholate (DC) and cholate (C) and their glycine (G) and taurine (T) conjugates) and LPC was determined using erythrocyte haemolysis as a model parameter. Washed human erythrocytes were incubated for 15-60 min at 20 degrees C with media buffered at pH 8, 7 and 6. Bile salt toxicity was shown to be a function of type, concentration, pH and contact time with the membrane. At pH 7 toxicity decreased in the order LPC greater than unconjugated dihydroxy salts (SDC and SCDC) greater than conjugated deoxycholates (SGDC and STDC) greater than conjugated chenodeoxycholates (SGCDC and STCDC) greater than unconjugated trihydroxy salt (SC) greater than conjugated trihydroxy salts (SGC and STC). Incubation with equimolar combinations of bile salts (SDC+SCDC; STCDC+SGDC; SDC+STDC) indicated that the resultant damage was an additive function of the damage induced by the individual bile salts.

QELS and X-ray study of two dihydroxy bile salt aqueous solutions

Previously, aqueous solutions containing one dihydroxy and one trihydroxy bile salt together with 0.8 M NaCl were studied by means of small-angle X-ray scattering (SAXS) and quasi-elastic light-scattering (QELS) techniques. It was observed that even a small amount of the bile salt forming smaller size aggregates (trihydroxy salt) inhibits the growth of the bile salt forming bigger size aggregates (dihydroxy salt), giving rise to an apparent mean hydrodynamic radius ( R h) value lower than that of the dihydroxy salt alone. This phenomenon occurred when sodium glycocholate and taurocholate were added to sodium glycodeoxycholate (NaGDC) and taurodeoxycholate (NaTDC) cylindrical aggregates, formed by trimers. In this paper observed SAXS data of a NaGDC aqueous electrolyte solution are reported and compared with the calculated ones by using the cylindrical aggregate model. The satisfactory agreement strongly supports the validity of the model. Moreover, aqueous solutions containing two dihydroxy salts (NaGDC and NaTDC), together with 0.8 M NaCl to minimize charge interactions, are investigated by QELS measurements that give information on the growth processes of NaGDC and NaTDC aggregates as a function of their relative concentration. The R h-value of a fixed amount of bile salt increases by adding the other bile salt, in agreement with the presence of two-component aggregates formed by random or block association of NaGDC and NaTDC trimers and in disagreement with the hypotheses of the growth inhibition and the formation of two one-component aggregates. X-ray and atomic emission spectroscopy measurements of fibers drawn from NaGDC + NaTDC solutions show that the fiber composition reflects with good approximation that of the solution. Under the same bile salt and NaCl concentrations and at the same temperature, the R h-values at high ionic strength of one-bile-salt solution follow the order NaGDC > NaTDC. Our results show that the R h-value of a 100 mM two-bile-salt solution (NaGDC + NaTDC) is intermediate between those of 100 mM NaGDC and NaTDC pure solutions. Moreover, R h-values of solutions with constant NaGDC + NaTDC total concentration (100 mM) versus NaGDC or NaTDC concentration are fitted by straight lines, thus indicating that the aggregate size linearly depends on NaGDC and NaTDC molar fractions. All these results indicate that NaGDC and NaTDC trimers seem to be interchangeable to some extent in the two-component aggregates that very probably are formed by means of the block association process.

Diffusivity Study of Dihydroxy−Trihydroxy Bile Salt Systems

Previously, structural units inferred from X-ray diffraction analysis of fibers, crystals, and concentrated aqueous solutions were identified as models of dihydroxy and trihydroxy bile salt micellar aggregates. Dimers andtheir multiples (mainly octamers and hexadecamers) or structures derived from 7/1 helices (formed by trimers) were proposed for trihydroxy salts sodium glycocholate (NaGC) and taurocholate (NaTC) or for dihydroxy salts sodium glycodeoxycholate (NaGDC) and taurodeoxycholate (NaTDC), respectively, on the basis of strong experimental evidences. However, the radii of the cylindrical structures derived from 7/1 helices were unknown. This paper deals with small-angle X-ray scattering (SAXS) measurements that have permitted to estimate the cylindrical aggregate radius of NaGDC (about 22 A), whereas that of NaTDC is very probably about 3 A longer. SAXS and quasi-elastic light-scattering (QELS) data have shown a linear increase of the apparent mean hydrodynamic radius (within the approximate range 40-70 A) with the height of NaGDC and NaTDC cylindrical aggregates, suggesting that the main interparticle correlation effects, ascribed to charge, van der Waals, and volume excluded interactions, are small or compensate each other. Moreover, aqueous solutions containing one dihydroxy and one trihydroxy bile salt (NaGDC and NaGC, NaTDC and NaTC, NaGDC and NaTC, NaTDC and NaGC) and 0.8 M NaCl have been studied. QELS measurements give information on the growing processes of NaGDC and NaTDC aggregates as a function of NaGC and NaTC concentration. Experimental data and calculations performed using the above-mentioned models support that a small amount of the bile salt forming smaller size aggregates (trihydroxy salt) inhibits the growth of the bile salt forming bigger size aggregates (dihydroxy salt), giving rise to smaller size aggregates. Reasonably, this phenomenon is due to the very different and not interchangeable structures of dihydroxy and trihydroxy bile salt aggregates. It seems also that the aggregate growth is a little more inhibited when the trihydroxy molecule polar head is different from that of the aggregate molecules. Trihydroxy salt monomers and oligomers compete with dihydroxy salt trimers in the growth process and interact with their cylindrical aggregates especially by means of polar forces.

Bile salt aggregates in the gas phase: an electrospray ionization mass spectrometric study.

Helical and ordered structures have previously been identified by X-ray diffraction analysis in crystals and fibers of bile salts, and proposed as models of the micellar aggregates formed by trimeric or dimeric units of dihydroxy and trihydroxy salts, respectively. These models were supported by the results of studies of micellar bile salt solutions performed with different experimental techniques. The study has now been extended to the gas phase by utilizing electrospray ionization mass spectrometry (ESIMS) to investigate the formation and the composition of aggregates stabilized by noncovalent interactions, including polar (ion-ion, ion-dipole, dipole-dipole, hydrogen bonding etc.) and apolar (van der Waals and repulsive) interactions. The positive and negative ESIMS spectra of sodium glycodeoxycholate (NaGDC), taurodeoxycholate (NaTDC), glycocholate (NaGC), and taurocholate (NaTC) aqueous solutions, recorded under different experimental conditions, show in the first place that aggregates analogous to those present in micellar solutions do also exist in the gas phase. Furthermore, consistently with the condensed-phase model, the positive-ion spectra show that the trimers are the most stable oligomers among the aggregates of dihydroxy salts (NaGDC and NaTDC) whilst the dimers are the most stable among the aggregates of trihydroxy salts (NaGC and NaTC). Moreover, the binding energy of the constituent glycocholate salt units in most gaseous oligomers exceeds that of the corresponding taurocholate units. The ESIMS evidence has been confirmed by vapor-pressure measurements performed on NaGC and NaTC crystals and NaGDC and NaTDC fibers, the results of which show that the evaporation enthalpy of glycocholate exceeds that of taurocholate by some 50 kJ mol(-1).

Interaction between bile salts and beta-adrenoceptor antagonists.

The interactions between beta-adrenoceptor antagonists and bile salts were investigated by microcalorimetry. Nadolol and oxprenolol interactions with dihydroxy salts could be described by a 1:1 interaction model with the thermodynamic parameters indicating that the drugs were incorporated within the bile salt aggregates. This weak interaction was primarily hydrophobic although electrostatic attraction also played a role. Atenolol and metoprolol did not interact with the dihydroxy salts. None of the compounds interacted with trihydroxy bile salts or with salts below their aggregation concentration. Phase separation resulted when propranolol and alprenolol were present in dihydroxy salt solutions above a certain concentration with the interaction being of a hydrophobic and electrostatic nature. The implications of these results on in-vivo drug absorption are discussed.

Interaction of bile salts with calcium hydroxyapatite: Inhibitors of apatite formation exhibit high–affinity premicellar binding

Of the major human bile salts, only the glycine-conjugated dihydroxy species prevent the transformation of amorphous calcium phosphate to calcium hydroxyapatite, a component of gallstones; we have proposed that this inhibition occurs by competition between the bile salt and HPO4(2-) anions for binding site on the apatite crystal embryo. Now we show that the binding affinity of bile salts to fully mature hydroxyapatite has the following order: glycine-conjugated dihydroxy salts > taurine-conjugated dihydroxy salts > glycocholate approximately taurocholate. Glycine-conjugated dihydroxy bile salts bound with high affinity as "premicellar" aggregates, but the remaining species appeared to bind as a wider range of aggregate sizes. Glycochenodeoxycholate binding was decreased as the pH increased from 6.6 to 9.8 and the apatite surface charge reversed from net positive to net negative. Binding was competitively inhibited by HPO4(2-), but not by H2PO4-. Ca2+ promoted the binding of glycochenodeoxycholate, taurochenodeoxycholate and glycocholate, and for the latter two bile salts the increase was associated with enhanced "premicellar" binding. The binding of taurocholate was not influenced by Ca2+. When either glycocholate or taurocholate was mixed with glycochenodeoxycholate, mixed aggregates were formed that had a lower affinity for apatite than had pure glycochenodeoxycholate aggregates. Because only glycine-conjugated dihydroxy bile salts inhibit apatite formation, these results suggest that inhibition depends on high-affinity "premicellar" bile salt-apatite binding.

Pruritic effect of bile salts.

The pruritic effect of purified bile salts has been tested by applying them to blister bases. All the salts tested were pruritogens, but the dihydroxy salts (especially unconjugated chenodeoxycholate) were more effective than the trihydroxy salts. This may explain the poor correlation between total serum bile salt concentration and pruritus in obstructive jaundice.