Deoxycholic Research Articles

Stepwise Aggregation of Cholate and Deoxycholate Dictates the Formation and Loss of Surface-Available Chirally Selective Binding Sites.

Bile salts are facially amphiphilic, naturally occurring chemicals that aggregate to perform numerous biochemical processes. Because of their unique intermolecular properties, bile salts have also been employed as functional materials in medicine and separation science (e.g., drug delivery, chiral solubilization, purification of single-walled carbon nanotubes). Bile micelle formation is structurally complex, and it remains a topic of considerable study. Here, the exposed functionalities on the surface of cholate and deoxycholate micelles are shown to vary from one another and with the micelle aggregation state. Collectively, data from NMR and capillary electrophoresis reveal preliminary, primary, and secondary stepwise aggregation of the salts of cholic (CA) and deoxycholic (DC) acid in basic conditions (pH 12, 298 K), and address how the surface availability of chirally selective binding sites is dependent on these sequential stages of aggregation. Prior work has demonstrated sequential CA aggregation (pH 12, 298 K) including a preliminary CMC at ca. 7 mM (no chiral selection), followed by a primary CMC at ca. 14 mM that allows chiral selection of binaphthyl enantiomers. In this work, DC is also shown to form stepwise preliminary and primary aggregates (ca. 3 mM DC and 9 mM DC, respectively, pH 12, 298 K) but the preliminary 3 mM DC aggregate is capable of chirally selective solubilization of the binaphthyl enantiomers. Higher-order, secondary bile aggregates of each of CA and DC show significantly degraded chiral selectivity. Diffusion NMR reveals that secondary micelles of CA exclude the BNDHP guests, while secondary micelles of DC accommodate guests, but with a loss of chiral selectivity. These data lead to the hypothesis that secondary aggregates of DC have an exposed binding site, possibly the 7α-edge of a bile dimeric unit, while secondary CA micelles do not present binding edges to the solution, potentially instead exposing the three alcohol groups on the hydrophilic α-face to the solution.

92 Increased Esophagogastric Junction Distensibility in GERD Patients Assessed with the Endoscopically Placed Functional Luminal Imaging Probe (Endoflip®)

Altered esophageal mucosa integrity (low electrical resistance and dilated intercellular spaces) may play a role in heartburn perception both in patients with non-erosive reflux disease and GERD patients refractory to PPI. Patients “on” PPI have gastric content with a higher pH (weakly acidic), bacterial overgrow and increased proportion of unconjugated bile acids. Weakly acidic solutions with the unconjugated bile acid deoxycholic (DC) disrupt esophageal mucosa integrity and induces dilated intercellular spaces (DIS) (Farre 2008). It is known that exposure of esophageal epithelial cells to unconjugated bile acids increases intracellular reactive oxygen species (ROS) and this effect is blocked by anti-oxidants. We aimed to further assess possible mechanisms by which weakly acidic reflux can provoke impairment of esophageal mucosa integrity in patients “on” PPI. We hypothesized that anti-oxidants may also prevent disruption of esophageal mucosa integrity induced by unconjugated bile acids in weakly acidic solutions. Methods: New Zealand rabbits were euthanized and segments from the esophageal mucosa were mounted in Ussing chambers. The luminal side was exposed for 30min to solutions containing 0.5, 2 and 5mM of DC and chenodeoxycholic acid (CDC) at pH 5.0. Transepithelial resistance (Rt) was measured for 90min. The antioxidants N-acetylcysteine (NAC) and vitamin C were added 30 min before exposure of the tissue to bile acids. After transepithelial resistance measurements, mucosa was assessed using transmission electron microscopy for diameter of intercellular spaces. Results: The unconjugated bile acids DC andCDC inweakly acidic conditions dose-dependently decreased Rt. At 5 mM the drop in Rt was 84.3±2.8 % and 75.3±2.1 % respectively (N=4, P<0.05). Application of NAC and vitamin C in the mucosal side (5, 10 and 20 mM), dose dependently attenuated the drop in Rt induced by DC (N=4). Application of anti-oxidants in the serosal side at the highest concentration did not produce any effect. Pretreatment with NAC 20mM prevented the dilation of intercellular spaces induced by DC (0.004±0.001μm vs. 0.048±0.004μm, N=3, P<0.05). Similar prevention was observed when antioxidants were applied before exposure to the unconjugated bile acid CDC.Conclusion: Esophageal mucosal damage induced by weakly acidic solutions with unconjugated bile acids, similar to reflux in GERD patients “on” PPI, can be prevented with anti-oxidants. Our experimental results suggest that anti-oxidants might be considered in the development of pharmachological strategies for patients with symptoms “on” PPI associated to persistent microscopic impairment of esophageal mucosa integrity.

Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage

Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 μM. Similarly, Hex (5–10 μM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2′-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer.

Self-assembled nanoribbons and nanotubes in water: energetic vs entropic networks

We present a comparative investigation of two opposite classes of self-assembled fibrillar networks. Ribbons and tubes having cross-sectional dimensions in the nanoscale can be formed in aqueous solutions of steroids derived, respectively, from deoxycholic (DC) and lithocholic (LC) acids. Rheological features distinguish energetic networks of DC ribbons rigidly fixed in cylindrical bundles and entropic transient networks of LC tubes weakly interacting in shear-sensitive suspensions. The two classes are characterized by their frequency sweep profiles, viscoelastic linear domains, scaling laws of the elastic shear modulus vs concentration, kinetics of formation of the networks, and their optical birefringence aspects. A theoretical context for networks of rigid fibers is used to account for the scaling exponents α in the G’ (and σ*) ∝C α laws (α=2.0 and 1.0, respectively, for DC and LC). The evolution observed in DC gels from ribbons to cylindrical fibers with monodisperse sections made up with four ribbons is an indication of an equilibrated balance between face-to-face attractions and untwisting elastic processes of the constitutive ribbons.

Lipophilicity of Selected Bile Acids as Determined by TLC. II. Investigations on RP18W Stationary Phase

The bile acids, such as: cholic (C), glycocholic (GC), glycodeoxycholic (GDC), chenodeoxycholic (CDC), deoxycholic (DC), lithocholic (LC), and glycolithocholic (GLC) acid were investigated with the use of reversed phase thin‐layer chromatography on RP18W (E. Merck, #1.14296), using methanol–water, organic mixture (acetonitrile–methanol 50:50, v/v)–water, acetone–water, dioxane–water in different volume compositions as a mobile phase. The chromatographic parameters of lipophilicity (R MW and ϕ0) of studied bile acids were determined. Lipophilic parameters (R MW and ϕ0) were compared both, with measured (log P exp), and calculated partition coefficients (A log P S, IA log P, log P KOWIN, x log P, c log P, log P Rekker). The most significant correlation was found between lipophilic parameters R MW and ϕ0 and log P KOWIN values. The results indicate that chromatographic R M values may be used as a measure of lipophilicity of the investigated bile acids.

Separation of Selected Bile Acids by TLC. II. One‐Dimensional and Two‐Dimensional TLC

The objective of this study was to describe a simple and efficient method to separate unconjugated bile acids and their glycine conjugates. The method described herein involves the use of a two‐dimensional thin layer chromatographic (2D TLC) technique. Seven bile acids: chenodeoxycholic (CDC), deoxycholic (DC), cholic (C), glycocholic (GC), lithocholic (LC), glycodeoxycholic (GDC), and glycolithocholic (GLC) were chromatographed on glass plates precoated with silica gel 60F254 and on aluminum plates precoated with silica gel 60, by developing them with: n‐hexane–ethyl acetate–acetic acid (25:20:5, v/v/v) in the first dimension and chloroform–n‐butanol–acetic acid–water (2:32:2:2, v/v/v/v) in the second dimension. These chromatographic conditions, using the 2D technique for bile acids separation, allowed for the complete separation of all the bile acids studied.

Deoxycholic (DOC) acid-induced expression of cyclooxygenase-2 (COX-2) in human colonic fibroblasts

Deoxycholic (DOC) acid-induced expression of cyclooxygenase-2 (COX-2) in human colonic fibroblasts

Deoxycholic (DOC) acid-induced expression of cyclooxygenase-2 (COX-2) in human colonic fibroblasts

Deoxycholic (DOC) acid-induced expression of cyclooxygenase-2 (COX-2) in human colonic fibroblasts

Gas liquid chromatography (GLC) analysis of hamster conjugated biliary bile acids

Many epidemological studies have suggested that elevated faecal bile acids are found in populations with a high incidence of colorectal cancer (1). Nevertheless case control studies have yielded conflicting results. Hill suggests that the anomalies between population and case control studies may be explained by a difficulty in matching cases with control groups in areas such as 'adenoma proneness' and large and small bowel ecology (2). Bile acids have been found to promote carcinogenesis in animal studies when given orally or intrarectally to animals exposed to specific colonic carcinogens (3,4). The rat has been used as a model for colorectal cancer by most investigators. This is not altogether satisfactory as several aspects of its biliary physiology and biochemistry differ markedly from the human. Rats possess a liver enzyme capable of 6-hydroxylation of the steroid nucleus producing muricholic acids (5). They also do not have a gallbladder capable of regulating biliary flow. HPLC studies of bile acids on hamster bile suggested that the hamster would probably be a better model for bile related human disease. The hamster has a gall bladder and does not 6-hydroxylate the steroid nucleus (6). Here we have used GLC to study in detail the conjugated biliary bile acids of the hamster and compared them to those of patients with benign gastrointestinal disorders. The patients were taken from the control arm of a colorectal cancer study. All had normal liver function tests. Hamster bile was collected from anaesthetised hamsters (Ketomine, Diazepam, Pentobarbitone) by cannulation of the common bile duct. Human bile was collected by endoscopy after the administration of IV CCK-PZ. Bile was immediately frozen and subsequently freeze dried prior to extraction. Bile acids were extracted with 3x21111 of ethanol, giving a recovery of 99.76% (SD=5.7, n=12). The taurine and glycine conjugated bile acids were separated as described by Setchell (5) and prepared for GLC analysis. The glycine and taurine fractions are the major fractions in bile and are the only ones reported here. Table 1 shows that the major bile acids in the hamster as in man are lithocholic (LC), deoxycholic (DC), chenodeoxycholic (CDC) and cholic acid (CA) in both the glycine and taurine fraction. The composition of human and hamster bile shows appreciable variation among the samples examined. The glycine and taurine ratio showed great variability, in the hamster g1ycine:taurine =4.52,CV-74% and in the human g1ycine:taurine =3.71, CV=62%. Statistical analysis showed that in bile the percentage of conjugated CA is significantly higher in the hamster (p<O.OOl,AOV) and conjugated CDC is significantly lower in the hamster (p<O.OOl,AOV). The per centages of conjugated LC and DC were not significantly different. we did not detect any of the muricholic bile acids in the hamster biles. TABLE 1 Mean % of total bile acids with S.D. Human Hamster Glycine Taurine Glycine Taurine LC 0.2520.61 0.8721.51 0.5520.59 0.4720.52 DC 11.6523.04 8.9223.33 17.9024.48 13.6125.09 CDC 20.5824.91 13.2725.69 35.1728.78 33.8527.77 CA 61.75211.59 46.8722.85 30.4424.68 34.2328.90 Sum 94.24 69.93 84.06 82.16

Effect of Bile Acids on Calcium Efflux from Isolated Rat Hepatocytes and Perfused Rat Livers

The changes in intracellular Ca2+ concentration [( Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGlu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.

Behaviour study of some cholic acids at the dropping mercury electrode using differential pulse polarography

The tensammetric behaviour of some unconjugated hydroxy derivatives [cholic (C) deoxycholic (DC). chenodeoxycholic (CDC) and ursodeoxycholic (UDC)] and conjugated ones [taurodeoxycholic (TDC) and glycodeoxycholic (GDC)] of cholanic acid at the dropping mercury electrode was studied by differential pulse polarography. The study showed that both the number and the position and configuration of hydroxyls, as well as the length of the lateral chain and the nature of the ionizable group, affect the adsorption properties of such compounds. The following adsorption order was determined for the compounds studied: TDC > GDC > C > DC > CDC > UDC.

Behaviour study of some cholic acids at the dropping mercury electrode using differential pulse polarography

The tensammetric behaviour of some unconjugated hydroxy derivatives [cholic (C). deoxycholic (DC). chenodeoxycholic (CDC) and ursodeoxycholic (UDC)] and conjugated ones [taurodeoxycholic (TDC) and glycodeoxycholic (GDC)] of cholanic acid at the dropping mercury electrode was studied by differential pulse polarography. The study showed that both the number and the position and configuration of hydroxyls, as well as the length of the lateral chain and the nature of the ionizable group, affect the adsorption properties of such compounds. The following adsorption order was determined for the compounds studied: TDC > GDC > C > DC > CDC > UDC.

Diabetes and intestinal cholesterol uptake from bile salt solutions.

A previously validated in vitro technique was used to determine the effect of diabetes mellitus on the intestinal uptake of cholesterol from various micellar bile salt solutions. The bile salts studied included cholic (C), taurocholic (TC), glycocolic (GC), chenodeoxycholic (CDC), taurochenodeoxycholic (TCDC), glycochenodeoxycholic (GCDC), deoxycholic (DC), taurodeoxycholic (TDC), and glycodeoxycholic (GDC). In control rats there was a reciprocal decline in cholesterol uptake with increasing concentrations of these nine bile acids, and cholesterol uptake was greater from the conjugated primary bile acids than from the unconjugated ones. With a 5 mM concentration of bile acids, the ratios of the uptake of 0.2 mM cholesterol in control rats were C = CDC = DC, TCDC greater than TC greater than TDC, and GC = GCDC greater than GDC; with 20 mM concentrations, the ratios of cholesterol uptake in control rats were C greater than CDC greater than DC, TC greater than TCDC greater than TDC, and GC = GCDC greater than GDC. In the diabetic animals cholesterol uptake was higher than in control rats when using 5 or 20 mM of each of the conjugated bile acids and with cholic acid. In contrast, cholesterol uptake was similar in diabetic and control animals when cholesterol was solubilized with 5 or 20 mM CDC or DC. These differences in cholesterol uptake using the various bile acids and the failure of CDC and DC to facilitate the enhanced uptake of cholesterol in diabetic animals remains unexplained.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of active and passive transport of bile acids in rabbit intestine

Previous studies have indicated that saturable, Na +-bile acid coupled transport is absent in the ileum throughout most of the suckling period and that the maturation of ileal bile acid uptake which occurs during weaning results primarily through an increase in functional bile acid carriers within the ileal brush border membrane. This study was undertaken in weanling and adult rabbits to establish the effect of maturation on the active ileal and passive jejunal and colonic uptake of 8 bile acids. The in vitro uptake of a range of concentrations of cholic (C), taurocholic (TC), glycocholic (GC), chenodeoxycholic (CDC), taurochenodeoxycholic (TCDC), glychochenodeoxycholic (GCDC), deoxycholic (DC) and taurodeoxycholic (TDC) acid was determined. Active ileal uptake of C, TC, GC, DC and TDC was greater in adult that in weanling animals, whereas uptake of CDC, TCDC and GCDC was similar in both groups. The relative permeability for passive uptake of bile acids into the jejunum and colon was similar in young and adult rabbits. Jejunal mucosal surface area was similar in the two groups, but was greater in the ileum in the adult than in the weanling rabbits due to an increase in villus height, width, and number of villi per unit serosal length. However, the age-associated differences in active bile acid uptake were not explained simply on the basis of these differences in ileal mucosal surface area or the concentration of bile acids in the intestinal lumen. Thus, maturation of the rabbit from weanling to adulthood does not influence the relative passive permeability of the jejunum or colon to bile acids, but does increase active ileal uptake of conjugated and unconjugated cholic acid and deoxycholic acid, but not chenodeoxycholic acid

Intestinal uptake of bile acids: Effect of external abdominal irradiation

Abdominal irradiation has recently been shown to influence the uptake of hexoses, amino acids, fatty acids and cholesterol into the jejunum of rats. The present studies were undertaken with a previously validated in vitro technique to determine the effect of abdominal irradiation from a cesium 137 source on the rates of uptake of six bile acids into the jejunum, ileum, and colon. In the ileum of control rats, there were marked differences in the value of the apparent Michaelis constant (Km ∗), maximal transport rate (J d m), and apparent passive permeability coefficient (Pd ∗) between cholic (C), glycocholic (GC), taurocholic (TC), chenodeoxycholic (CDC), and glycochenodeoxycholic (GCDC), and deoxycholic (DC) acid. The Km ∗ for each bile acid except. DC was lower three and 14 days after 600 rad, whereas the J d m for GC fell, but rose for TC, CDC, GCDC and DC and was unchanged for C. The Pd ∗ rose for C, GC, and DC, fell for TC and CDC, but remained unchanged for GCDC 14 days after irradiation. After 600 rad the value of Pd ∗ in the colon was increased at day 3 and 14 for CDC and GCDC, but was unchanged for GC and TC and was decreased for C. The uptake of bile acids was also affected by 300 rad and by 900 rad, but the direction and magnitude of the change was influenced by the intestinal site, the dose of irradiation, and the type of bile acid. The results show that: 1) there likely are multiple ileal carriers for bile acids; 2) abdominal irradiation has a variable effect on these carriers; 3) the passive permeability to bile acids varies with the bile acid and with the site along the intestine; and 4) abdominal irradiation is associated with a rise in the colonic permeability to only some bile acids.

Uptake of bile acids into rat intestine. Effect of diabetes mellitus.

Diabetes mellitus is associated with the enhanced uptake of several nutrients. The purpose of this study was to use an established in vitro technique to examine the uptake of bile acids into jejunum, ileum, and colon of control (CON) and streptozotocin-diabetic rats (DM). When the bulk phase was stirred to reduce the effective thickness of the unstirred layer, the ileal uptake of 1.5-15 mM cholic (C), glycocholic (GC), taurocholic (TC), chenodeoxycholic (CDC), glycochenodeoxycholic (GCDC), and deoxycholic (DC) acid was similar in CON and DM. The relative values of the maximal transport rates (Jdm) were CDC greater than GC greater than GCDC greater than C greater than TC = DC, and similar relative values were observed for the Michaelis constants (Km). The values of Jdm and Km for each bile acid were similar in CON and in DM. In CON and at pH 7.4 an inverse linear relationship was noted between the number of hydrogen bonds in the bile acids and the natural logarithm of the permeability coefficient (Pd) times the square root of the molecular weight of the bile acid. This slope reflected the incremental change in free energy, delta delta Fw leads to I, associated with the uptake of bile acids; the value of delta delta Fw leads to I was similar for jejunum, ileum, and colon of CON, but was lower in jejunum of DM than CON. Thus, DM is associated with a greater relative permeability of the jejunum but not the ileum or the colon to a series of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from clostridium absonum ATCC# 27555 and cellular response of this organism to bile acid inducers.

Both 7 alpha- and 7 beta-hydroxysteroid dehydrogenases (HSDH) were induced by either chenodeoxy-(CDC) or deoxycholic (DC) acid in C. absonum. 7 beta-HSDH was partially purified 35-fold from CDC-induced cultures of C. absonum by Procion Red (PR) affinity chromatography and high performance liquid chromatography (HPLC) using a TSK 3000 SW gel filtration column. A relative molecular weight of 200 K was estimated for 7 beta-HSDH using Sephacryl S-300 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the 35-fold purified 7 beta-HSDH showed six polypeptides in the molecular weight range of 40-50 K. Induction of cultures of C. absonum with CDC or DC (0.4 mM) also resulted in the differential synthesis of at least five new polypeptides with molecular weights of 94 K, 42 K, 32 K, 21 K, and 16 K. The 16 K polypeptide was induced by DC but not by CDC. SDS-PAGE of Triton X-100-solubilized membranes from these extracts revealed the presence of a new membrane-associated polypeptide of molecular weight 80 K. The soluble inducible polypeptides were eliminated during purification of the 7 alpha- and 7 beta-HSDH and, therefore, are not required for these enzyme activities. It is proposed that this organism synthesized 7 alpha- and 7 beta-HSDH as well as a series of other proteins in response to bile acids which may, in the absence of the dehydrogenases, be toxic to C. absonum. The HSDH's catalyze the epimerization of chenodeoxycholic acid to ursodeoxycholic acid, which is less toxic than the chenodeoxycholic acid. The other proteins may assist the survival of the organism in a high bile acid environment by mechanisms not yet understood.

Serum and urinary bile acids in patients with primary biliary cirrhosis.

Serum concentrations and daily urinary excretions of unsulphated and total cholic (C) and chenodeoxycholic (CDC) acid were determined by radioimmunoassay in 15 patients with primary biliary cirrhosis. Thirteen patients had increased fasting serum bile acid concentrations; two of them had an increase of C only. An increase of C and CDC in serum was always followed by an increased urinary excretion of C and CDC. The individual serum bile acids were separated by gas-liquid chromatography. 3 beta-hydroxy-5-cholenoic acid was increased in nine patients, and its serum concentration was correlated to the total serum bile acid concentration. Deoxycholic (DC) and lithocholic (LC) acid were found in most patients, but their serum concentrations were not correlated to the total serum bile acid concentration. Minor bile acids comprised an average of 2% of the total concentration.