Solubility of calcium salts of unconjugated and conjugated natural bile acids.

The approximate solubility products of the calcium salts of ten unconjugated bile acids and several taurine conjugated bile acids were determined. The formation of micelles, gels, and/or precipitates in relation to Ca2+,Na+, and bile salt concentration was summarized by "phase maps." Because the ratio of Ca2+ to bile salt in the precipitates was ca. 1:2, and the activity of Ca2+ but not that of bile salt (BA-) could be measured, the ion product of aCa2+ [BA-]2 was calculated. The ion product (= Ksp) ranged over nine orders of magnitude and the solubility thus ranged over three orders of magnitude; its value depended on the number and orientation of the hydroxyl groups in the bile acid. Ion products (in units of 10(-9) mol/l)3 were as follows: cholic (3 alpha OH,7 alpha OH,12 alpha OH) 640; ursocholic (3 alpha OH,7 beta OH,12 alpha OH) 2300; hyocholic (3 alpha OH,6 alpha OH,7 alpha OH) 11; ursodeoxycholic (3 alpha OH,7 beta OH) 91; chenodeoxycholic (3 alpha OH,7 alpha OH) 10; deoxycholic (3 alpha OH,12 alpha OH) 1.5; 12-epideoxycholic (lagodeoxycholic, 3 alpha OH,12 beta OH) 2.2; hyodeoxycholic (3 alpha OH,6 alpha OH) 0.7; and lithocholic (3 alpha OH) 0.00005. The critical micellization temperature of the sodium salt of murideoxycholic acid (3 alpha OH,6 beta OH) was greater than 100 degrees C, and its Ca2+ salt was likely to be very insoluble. Taurine conjugates were much more soluble than their corresponding unconjugated derivatives: chenodeoxycholyltaurine, 384; deoxycholyltaurine, 117; and cholyltaurine, greater than 10,000. Calcium salts of unconjugated bile acids precipitated rapidly in contrast to those of glycine conjugates which were metastable for months. Thus, hepatic conjugation of bile acids with taurine or glycine not only enhances solubility at acidic pH, but also at Ca2+ ion concentrations present in bile and intestinal content.     

The effect of bile salts and calcium on isolated rat liver mitochondria

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.     

Bifidobacteria and probiotic effects: Action of Bifidobacterium species on conjugated bile salts

The effect of six different conjugated bile salts (two trihydroxyconjugated bile salts: tauro and glycocholic acids; and four dihydroxyconjugated bile salts: tauro- and glycochenodeoxycholic, tauro- and glycodeoxycholic acids) on eight bifidobacteria strains were studied. A strong growth-inhibitory effect was observed (80% at 0.95mm) for each bile salt and strain. This phenomenon was explained by the production of deconjugated bile salt during bifidobacteria growth. The deconjugation phenomenon was concurrent with biomass production, and deconjugated bile salts were the sole compound produced during bifidobacteria biotransformation. In resting cell experiments, differences appeared between the strains and the kind of bile salts, particularly concerning taurocholic acid. The Bifidobacterium longum strains were the most efficient among the bacteria tested.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

Concentration-dependent effects of sodium cholate and deoxycholate bile salts on breast cancer cells proliferation and survival

Bile acids (BAs) are bioactive molecules that have potential therapeutic interest and their derived salts are used in several pharmaceutical systems. BAs have been associated with tumorigenesis of several tissues including the mammary tissue. Therefore, it is crucial to characterize their effects on cancer cells. The objective of this work was to analyse the molecular and cellular effects of the bile salts sodium cholate and sodium deoxycholate on epithelial breast cancer cell lines. Bile salts (BSs) effects over breast cancer cells viability and proliferation were assessed by MTS and BrdU assays, respectively. Activation of cell signaling mediators was determined by immunobloting. Microscopy was used to analyze cell migration, and cellular and nuclear morphology. Interference of membrane fluidity was studied by generalized polarization and fluorescence anisotropy. BSs preparations were characterized by transmission electron microscopy and dynamic light scattering. Sodium cholate and sodium deoxycholate had dual effects on cell viability, increasing it at the lower concentrations assessed and decreasing it at the highest ones. The increase of cell viability was associated with the promotion of AKT phosphorylation and cyclin D1 expression. High concentrations of bile salts induced apoptosis as well as sustained activation of p38 and AKT. In addition, they affected cell membrane fluidity but not significant effects on cell migration were observed. In conclusion, bile salts have concentration-dependent effects on breast cancer cells, promoting cell proliferation at physiological levels and being cytotoxic at supraphysiological ones. Their effects were associated with the activation of kinases involved in cell signalling.

     

Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed greater than 99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.     

Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin-Harkins plots. The degree of counterion binding was found to be 0.37-0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I1/I3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC approximately = NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.     

Isolation and identification of bile salts conjugated with cysteinolic acid from bile of the red seabream, Pagrosomus major.

Bile salts present in gallbladder of wild and cultured red seabream, Pagrosomus major, a marine teleost were analyzed. The bile from wild red seabream was found to contain two previously unknown bile salts along with two known bile salts, taurocholate and taurochenodeoxycholate. Isolation of each bile salt was performed by column chromatography. Fast atom bombardment mass spectra of the unknown bile salts showed the molecular ions (M-H)- of m/z 544 and 528 which are shifted 30 mass units upfield compared to those (m/z 514 and 498) of taurocholate and taurochendeoxycholate, respectively; this is consistent with the presence of cysteinolic acid (mol wt 155) instead of taurine (mol wt 125). Enzymatic hydrolysis of the bile salts released cholic acid and chenodeoxycholic acid, respectively, and an amino acid that was identified as D-cysteinolic acid by direct comparison with an authentic sample. From these results, the bile salts in the bile of wild red seabream were identified as the conjugates of cholic acid and chenodeoxycholic acid with cysteinolic acid. 1H- and 13C-magnetic resonance spectra of the bile salts were also consistent with the proposed structure. The cysteinolic acid conjugates were found only in wild and not in cultured red seabream; this distinction seems to result from differences in dietary cysteinolic acid.     

Aggregation patterns of bile salts: crystal structure of calcium cholate chloride heptahydrate

Crystals of calcium cholate chloride heptahydrate, CaC24H39O7Cl . 7H2O, are monoclinic, space group P2(1), with a = 11.918(2), b = 8.636(1), c = 15.302(3) A, beta = 97.93(3) degrees, V = 1559.9(8) A3, and Z = 2. A trial structure was obtained by Patterson and Fourier techniques and was refined by full-matrix least-squares calculations using absorption corrected CuK-alpha diffractometer data. The final R index is 0.047. The crystal structure contains bilayer-type arrangements, with hydrophobic portions of cholate rings sandwiched between layers of polar groups that are interacting with calcium ions and water molecules. The calcium ion is coordinated to five water molecules and to the two carboxylate oxygen atoms of the cholate residue. Two additional water molecules are involved only in crystal packing through the formation of hydrogen bonds. Cholate-cholate hydrophobic interactions involve contacts between the hydrocarbon portions of the carboxylate sidechains and the A and B rings. This results in a staggered packing pattern that is nearly identical to that found in crystals of sodium cholate and rubidium deoxycholate. Similar bilayer aggregation patterns may also be involved in the formation of bile salt micelles in aqueous media. The characteristic bilayer packing arrangement can accommodate a variety of cation-binding patterns, as evidenced by the finding that calcium, sodium, and rubidium ions interact with the polar faces of the bilayers in different ways. The carboxylate sidechain displays two different conformations in the crystal structure of calcium cholate chloride heptahydrate. Variation in sidechain conformation may be of importance in the adjustment required to accommodate different cation coordination schemes.     

Structural and kinetic studies on the solubilization of lecithin by sodium deoxycholate.

Mixed dispersions of egg phosphatidylcholine (PC) and the bile salt sodium deoxycholate (DOC) were prepared by various methods, and their turbidities and proton magnetic resonance spectra were studied as a function of time. The spectra of dispersions prepared by dissolving both components in a common organic solvent and replacing the organic solvent by water did not change with time, indicating that the mixed aggregates formed represent "a state of equilibrium". In the 1H NMR spectra of these mixed aggregates, only signals from small mixed micellar structures were narrow enough to be observed. The dependence of the NMR line widths on the molar ratio of DOC to PC (R) is interpreted in terms of a model for the PC--DOC mixed micelles, according to which PC is arranged as a curved bilayer, the curvature of which increases with increasing R. Upon mixing PC with aqueous solutions of DOC, we found that the mixed aggregates formed are slowly reorganized and ultimately reach the same state of equilibrium. This reorganization was found to be a pseudo-first-order process, the rate constant of which depends linearly upon the detergent concentration. This process involves saturation of the outer bilayers of the multilamellar PC by detergent, followed by transformation of these bilayers into mixed micelles. It is concluded that the solubilization occurs through consecutive "peeling off" of lecithin bilayers.