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

Abstract

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.

Biomimetic Gallstone Formation: Crystallization of Calcium Carbonate by the Evolving Taurocholate−Lecithin−Cholesterol Complex Lipid System

“…Gallstone is a composite material of organic and inorganic components. Much of the attention to gallstone formation has still been paid to the prerequisites of the other organic components and complex fluid structures in gallbladder bile, including cholesterol and its solubilization, − bile salt micellar aggregation, − phospholipid vesiculation, − cholesterol−phospholipid vesiculation, − bile salt−phospholipid mixed micelle formation, − and bile salt−cholesterol−phospholipid mixed vesiculation. , Yet, there are a few papers addressing the contributions , of calcium ions and calcium carbonate phase on cholesterol nucleation, , calcium carbonate−cholesterol interactions, , calcium−phospholipid bindings, and calcium−bile salts interactions. − …”

“…443.92 mg of CaCl 2 were added into the first 20 mL and 336 mg of NaHCO 3 were added into the second 20 mL of model bile solution to yield 20 mL of 0.2 M CaCl 2 and 20 mL of 0.2 M NaHCO 3 solutions at 22 °C. A small volume of Tris buffered saline was added to both 20 mL portions of solution to achieve a pH ∼7.5. ,, CaCO 3 crystals began to form when the two bottles were mixed and stirred at 750 rpm. The final concentrations of taurocholate, lecithin, and cholesterol in a 40 mL aqueous solution were 0.0839, 0.0289, and 0.0107 M, respectively.…”

“…A small volume of Tris buffered saline was added to both 20 mL portions of solution to achieve a pH ∼7.5. 23,61,71 CaCO 3 crystals began to form when the two bottles were mixed and stirred at 750 rpm. The final concentrations of taurocholate, lecithin, and cholesterol in a 40 mL aqueous solution were 0.0839, 0.0289, and 0.0107 M, respectively.…”

“…53,54 Yet, there are a few papers addressing the contributions 23,55 of calcium ions and calcium carbonate phase on cholesterol nucleation, 25,56 calcium carbonate-cholesterol interactions, 57,58 calcium-phospholipid bindings, 59 and calcium-bile salts interactions. [60][61][62] Although calcium carbonate is known to have three crystalline polymorphs, 63 vaterite, aragonite, and calcite, the common type of cholesterol-rich, nonpigmented gallstones 55,[64][65][66][67] contain two polymorphs of calcium carbonate: spheroliths of the thermodynamically unstable vaterite and stable calcite, mixed with about 70 wt % of broken sheets of cholesterol monohydrate 68 microcrystals. These findings raise a number of interesting questions.…”