Concentration Of Cholic Acid Research Articles

Antifungal evaluation of cholic acid and its derivatives on Candida albicans by microcalorimetry and chemometrics

In the last few years, several fungus infections caused by multidrug-resistant pathogenic agents have got tremendous emergence and prevalence. Screening for novel antifungal agents is in great demand, but traditional microbiological techniques are far from sufficient to meet that requirement. In this study, a non-invasive and non-destructive microcalorimetric method was performed to investigate the antifungal activities of cholic acid (CA) and its derivatives, glycocholic acid (GCA) and taurocholic acid (TCA) on the multiplying and non-multiplying metabolism of Candida albicans. Then, the heat-flow power-time curves of C. albicans growth affected by different concentrations of CA, GCA and TCA were studied by similarity analysis (SA), the quantitative thermokinetic parameters from these curves were analyzed by multivariate analysis of variance (MANOVA) and principal component analysis (PCA). By comparing the values of two main parameters, P 2 (the heat-flow output power of the highest peak) and Q 2 (the heat output of the second exponential growth phase) of C. albicans, it could be found that CA had the strongest antifungal activity among the three steroid compounds, which might be used as a potential antifungal agent in the future. This study provided a useful method and idea of microcalorimetry with chemometrics to efficiently evaluate the antifungal activities of bile acid derivatives, giving some references for screening out new antifungal agents. However, it has to be stressed that all these experiments are carried out in vitro and they still require clinical validation.

Fasting plasma chenodeoxycholic acid and cholic acid concentrations are inversely correlated with insulin sensitivity in adults

BackgroundAccumulating data suggest a novel role for bile acids (BAs) in modulating metabolic homeostasis. BA treatment has been shown to improve glucose tolerance and to increase energy expenditure in mice. Here, we investigated the relationship between fasting plasma BAs concentrations and metabolic parameters in humans.FindingsFasting plasma glucose, insulin and lipid profile were measured in 14 healthy volunteers, 20 patients with type 2 diabetes (T2D), and 22 non-diabetic abdominally obese subjects. Insulin sensitivity was also assessed by the determination of the glucose infusion rate (GIR) during a hyperinsulinemic-euglycemic clamp in a subgroup of patients (9 healthy and 16 T2D subjects). Energy expenditure was measured by indirect calorimetry. Plasma cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) concentrations were analyzed by gas chromatograph-mass spectrometry. In univariable analysis, a positive association was found between HOMA-IR and plasma CDCA (β = 0.09, p = 0.001), CA (β = 0.03, p = 0.09) and DCA concentrations (β = 0.07, p < 0.0001). Spearman analysis retrieved an inverse relationship between plasma CDCA (r = -0.44, p = 0.03), CA (r = -0.65, p = 0.001) and the GIR. HOMA-IR remained positively associated with CDCA (β = 0.11, p = 0.01), CA (β = 0.04, p = 0.01) and DCA (β = 0.06, p = 0.007) in multivariable analysis, after adjustment for age, gender, BMI, HbA1C and plasma lipid parameters. In contrast, HbA1c, energy expenditure and plasma lipid concentrations were not correlated with plasma BAs levels in multivariable analysis.ConclusionsBoth plasma CDCA, CA and DCA concentrations were negatively associated with insulin sensitivity in a wide range of subjects.

Influence of Cholic Acid on Phase Transition, Rheological Behavior, and Microstructures of Salt-Free Catanionic Surfactant Mixtures

The influence of a biosurfactant, cholic acid (CA), on the phase transition, microstructure, and rheological properties of two salt-free catanionic surfactant systems, (i) tetradecyltrimethylammonium laurate (TTAL) vesicular solution and (ii) a mixture solution of tetradecyltrimethylammonium hydroxide (TTAOH)/lauric acid (LA)/H(2)O, was investigated. The TTAL vesicular system remains homogeneous at 25 degrees C up to 9.8 mmol.L(-1) CA. At low concentrations of CA, the addition of CA causes a decrease in the viscosity of TTAL vesicular solutions. Upon further addition, the viscosity increases. When LA is gradually substituted by CA in the TTAOH/LA/H(2)O system with equimolar TTAOH and LA to form the TTAOH/(CA + LA)/H(2)O system, the influence of the molar fraction of CA, x = n(CA)/(n(LA) + n(CA)), on the phase transition and rheological behavior was also investigated. With increasing x, the system changes from a birefringent L(alpha) phase of equimolar TTAOH and LA into an L(1)/L(alpha) double-phase, and a single L(1) phase. In the birefringent L(alpha) phase of x = 0.2, cryo-TEM observations demonstrate that vesicles and tube-like and branched tube structures coexist, indicating that the unique structure of CA has a significant influence on the phase transformations of the mixtures. At higher x value, x = 0.5, a dilute solution was obtained, with a lower viscosity comparable to water. However, at x from 0.25 to 0.5, the system exhibits characteristic worm-like micelles with shearing thinning behavior at low and high shear rates but a Newtonian fluid behavior at intermediate shear rate range. At x = 0.2, the sample separates into two phases. The upper phase also shows shear thinning behavior and a narrow plateau at intermediate shear rate. At x < 0.2, the system exhibits similar rheological behavior to the original TTAL vesicular solution. For comparison, an investigation of the cetyltrimethylammonium hydroxide (CTAOH)/LA/H(2)O system with long chain cationic surfactant is also reported. The results of these studies may provide a better understanding of biological membranes and the effects of biosurfactants on surfactant phase transitions.

Plasma Bile Acid Concentrations in Patients with Human Immunodeficiency Virus Infection Receiving Protease Inhibitor Therapy: Possible Implications for Hepatotoxicity

To evaluate whether patients with human immunodeficiency virus (HIV) infection who were receiving protease inhibitor therapy had altered bile acid concentrations compared with noninfected control subjects, and whether bile acid concentrations could predict the onset of hepatotoxicity caused by protease inhibitors. Retrospective sample analysis from a prospectively conducted clinical trial. Academic research center. Eleven adults with advanced HIV disease who were taking protease inhibitor-based antiretroviral therapy, one of whom had developed protease inhibitor-induced hepatotoxicity. Plasma concentrations of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and taurocholic acid (TC) were analyzed by using a novel high-performance liquid chromatography with tandem mass spectrometry detection method. Comparisons of the relative contribution of each bile acid to the total bile acid pool were made with previously published values and with bile acid concentrations contained in two pooled plasma samples from healthy, non-HIV-infected volunteers analyzed in our laboratory. Each pooled plasma sample used for this analysis contained contributions from three non-HIV-infected volunteers. The LCA and TC concentrations in HIV-infected patients were 3-4-fold higher than those previously reported for non-HIV-infected subjects; concentrations of other bile acids were similar to those of previous reports. The relative contribution of CDCA to the total bile acid pool was 9% in HIV-infected patients compared with 30-50% in noninfected subjects. Total and individual bile acid concentrations in the HIV-infected patient who developed hepatotoxicity were similar to the bile acid concentrations from the other study patients who did not develop hepatotoxicity. These data suggest that bile acid concentrations may be altered by HIV infection and/or protease inhibitor therapy. However, further investigations should be performed to assess whether antiretroviral-associated hepatotoxicity can be predicted by alterations in individual bile acid concentrations.

Effect of SLCO1B1 polymorphism on the plasma concentrations of bile acids and bile acid synthesis marker in humans

Organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) is a sinusoidal influx transporter of human hepatocytes. Our aim was to characterize the role of OATP1B1 in the hepatic uptake of bile acids in vivo. Fasting blood samples were drawn from 24 healthy volunteers with SLCO1B1 c.388AA-c.521TT (*1A/*1A) genotype, eight with c.388GG-c.521TT (*1B/*1B) genotype, 24 with c.521TC genotype, and nine with c.521CC genotype. Plasma concentrations of 15 endogenous bile acids, their synthesis marker, and cholesterol were determined by liquid chromatography-tandem mass spectrometry. The concentrations of ursodeoxycholic acid, glycoursodeoxycholic acid, chenodeoxycholic acid, and glycochenodeoxycholic acid were approximately 50-240% higher in individuals with the SLCO1B1 c.521CC, c.521TC, or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), with the largest differences seen between the c.521CC and c.388GG-c.521TT individuals. The concentration of tauroursodeoxycholic acid was approximately 120% higher in individuals with the c.521TC genotype and that of taurochenodeoxycholic acid 110% higher in individuals with the c.521CC or c.521TC genotype than in those with the c.388GG-c.521TT genotype (P<0.05). The cholic acid concentration was approximately 30% higher in individuals with the c.521CC or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), but its conjugates remained unaffected by the genotype. The bile acid synthesis marker 7alpha-hydroxy-4-cholesten-3-one/cholesterol concentration ratio was 62 or 45% higher in the c.388AA-c.521TT participants than in the c.388GG-c.521TT or c.521TC participants, respectively (P<0.05). SLCO1B1 polymorphism considerably affects the disposition of several endogenous bile acids and bile acid synthesis marker, indicating that OATP1B1 plays an important role in the hepatic uptake of bile acids in vivo in humans.

The effect of cholic acid treatment on the oxidative status of soybean plants

The objective of this work was to study the effect of treatment of young soybean plants with cholic acid of different concentrations on their oxidative status. Young soybean plants, grown hydroponically for two weeks, were treated by adding cholic acid to the nutrient solution at the concentrations 20, 40, 60 and 80 mg/L, the control being without cholic acid. After one week, several parameters of the oxidative status were determined in the leaves and roots of the plants: contents of superoxide (-O?2 ), hydroxyl radicals (?OH) and glutathione (GSH), lipid peroxidation (LP), the superoxide dismutase (SOD) activity and the soluble protein accumulation, as well as the contents of chlorophylls and carotenoids. Treatments with cholic acid increased - O?2, LP, ?OH and GSH in the leaves of the treated plants, while only the OH content increased in the roots at higher cholic acid concentrations. The obtained results support the idea that cholic acid, as an elicitor of defense responses in plants, might act through the generation of an oxidative burst.

Gas chromatography–mass spectrometry of the trimethylsilyl (oxime) ether/ester derivatives of cholic acids: Their presence in the aquatic environment

This paper presents a derivatization, mass fragmentation study relating to the most common six cholic acids, such as cholic, lithocholic, chenodeoxycholic, ursodeoxycholic, 3-hydroxy,7-ketocholanic and dehydrocholic acids, identified and quantified as pollutants in the aquatic environment at the first time. Derivatizations have been performed with the two-step process (1: oximation, 2: silylation) varying the time and temperature of both reactions. Optimum responses have been obtained after 30 min oximation with hydroxylamine·HCl and 90 min silylation with hexamethyldisilazane and trifluoroacetic acid at 70 °C. Fragmentation patterns of the trimethylsilyl (oxime) ether/ester derivatives of all six cholic acids provided the theoretically expected, fully derivatized compounds. Reproducibility/linearity of derivatives calculated on the basis of the corresponding selective fragment ions, characterized by the relative standard deviation percentages of measurements, proved to be ≤4.9 (RSD%). The practical utility of the method was shown by the identification and quantification of cholic acids as pollutants in the aquatic environment. Subsequently to a solid phase extraction study varying the pH of extractions (pH 2, pH 4 and pH 7), applying the OASIS cartridges, it has been confirmed that the recoveries for all six cholic acids are acceptable, varying between 77% and 104%, and are independent on the pH. The total cholic acid content of a Hungarian wastewater plants’ influent wastewater varied between 184 μg/L and 356 μg/L, while the Danube rivers’ cholic acid content was 4.1 μg/L, only.

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

Direct and label-free detection of cholic acid based on molecularly imprinted photonic hydrogels

Here we report the design and preparation of a novel self-reporting sensor for cholic acid, an important biological compound produced in hepatocytes. Traditional detection methods of cholic acid are mostly dependent on analytical equipment, and are either time-consuming or require a derivatization process. In this work, a new approach based on molecularly imprinted photonic hydrogels (IPHs) is described, by which direct, sensitive and label-free detection of cholic acid can be achieved without any derivatization treatment and expensive instruments. The unique 3D ordered porous hydrogels that reveal optical changes in responsive to cholic acid concentration were prepared by combining colloidal crystal templating with the molecular imprinting technique. Due to their special hierarchical porous structure, which consists of 3D-ordered interconnected macroporous arrays with nanocavities derived from molecular imprinting, the formed photonic hydrogels allow rapid and ultrasensitive detection of the target analyte. The interconnected macropores are favorable for the rapid transport of the analyte in the hydrogel, while the inherent high affinity of nanocavities distributed in thin hydrogel walls allows IPHs to recognize the analyte with high specificity.

Effect of cholic acid and its keto derivatives on the analgesic action of lidocaine and associated biochemical parameters in rats

This study examined the effect of the structure and concentration of cholic acid and its keto derivatives on the local analgesic action of lidocaine in rats, measured by an analgesimetric method. The increase in bile acid concentrations in the administered lidocaine solution increased the duration of local anesthesia. It was found that the introduction of keto groups into the cholic acid molecule yielded derivatives with lower promotory action, i.e. decreased the duration of local anesthesia. The biochemical parameters investigated indicated that the keto derivatives of cholic acid exhibited no toxicity compared to that of cholic acid itself.

Determination of critical micellar concentrations of cholic acid and its keto derivatives

The critical micellar concentration (CMC) values of keto derivatives of cholic acid (3α,12α-dihydroxy-7-oxo-5β-cholanoic acid, 3α,7α-dihydroxy-12-oxo-5β-cholanoic acid, 12α-hydroxy-3,7-dioxo-5β-cholanoic acid, 3α-hydroxy-7,12-dioxo-5β-cholanoic acid, 3,7,12-triketo-5β-cholanoic acid) and cholic acid itself, were determined. Replacement of hydroxyl groups in cholic acid molecule with keto groups yields the derivatives whose CMC values increase with increase in the number of keto groups introduced. The CMCs of derivatives with the same number of keto groups but at different positions do not differ significantly. The relationship between the number of keto groups in the molecule of cholic acid keto derivatives and CMC value can be described by the following equation: CMC = 43 number of keto groups + 14.667. The effect of NaCl concentration on CMC increases with increase in the number of keto groups.

Mechanism of Growth Inhibition by Free Bile Acids in Lactobacilli and Bifidobacteria

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.

In VivoAntihepatotoxic Effects ofLigularia fischerivar.spiciformisand the Identification of the Active Component, 3,4-Dicaffeoylquinic Acid

Pretreatment with a methanolic extract of Ligularia fischeri var. spiciformis (Compositae) herb inhibited hepatotoxicities caused by CCl4, D-galactosamine (GalN), alpha-naphthylisothiocyanate (ANIT), and DL-ethionine in rats. An ethyl acetate (EtOAc) extract fractionated from the methanolic extract showed a strong inhibitory effect. A major component, 3,4-dicaffeoylquinic acid (DCQA), isolated from the methanolic extract was examined for antihepatotoxicity. Pretreatment with DCQA (5 and 10 mg/kg, p.o.) significantly reduced serum aminotransferases (alanine and aspartate), sorbitol dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, and lactate dehydrogenase activities during CCl4- or GalN-induced hepatotoxicity, suggesting that DCQA is a major principle for the antihepatotoxic activity of L. fischeri var. spiciformis. DCQA also partially restored bile flow and reduced total bilirubin and cholic acid concentrations in rats with ANIT-induced cholestasis. Treatment with DCQA inhibited the increase in triglyceride, cholesterol, and total lipids in DL-ethionine-induced fatty liver. These results support the traditionally held belief that this plant can be used for the treatment of jaundice and hepatic failure.

Effect of tibolone on turnover of cholesterol to bile acids in ovariectomized rats

Tibolone is used in breast cancer therapy in women. The most important aspect of this therapy concerns its influence on bone and lipid metabolism. Several studies have reported the effect of tibolone on plasma lipids. Approximately 40% of total cholesterol removal occurs by its conversion to bile acids, a process that takes place in the liver. The aim of the study was to evaluate the influence of tibolone on the conversion of cholesterol to bile acids in estrogen-deficient rats. Thirty female Wistar rats were divided into three groups: sham-operated controls, ovariectomized controls, and ovariectomized rats treated with tibolone. After 42 days of drug administration, bile was collected under anesthesia after administration of radioactive 4-C cholesterol. Bile was assayed for total C radioactivity; C bile acids were determined after thin-layer chromatography separation by using the isotopic technique. The ovariectomy decreased the concentrations of C-radioactive bile, taurocholic acid, and cholic acid. The administration of tibolone significantly increased the excretion of C-radioactive bile and C-radioactive bile acids as compared with ovariectomized controls. Moreover, the therapy with tibolone significantly increased the concentrations of C taurocholic acid, C glycocholic acid, cholic acid, and taurochenodeoxycholic plus taurodeoxycholic acids. The results of the present study suggest that tibolone may increase the concentrations of cholesterol and trihydroxy bile acids in bile. These changes may be associated with an increased risk of gallstone generation.

Complementary Stimulation of Hepatobiliary Transport and Detoxification Systems by Rifampicin and Ursodeoxycholic Acid in Humans

Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes

Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.

Effect of candidate vaginally-applied microbicide compounds on recognition of antigen by CD4+ and CD8+ T lymphocytes.

Vaginally applied antimicrobial compounds (microbicides) are being developed as an alternative method for preventing the spread of sexually transmitted diseases. In addition to identifying compounds effective against a spectrum of sexually transmitted pathogens, it will be important to ensure that these compounds are safe. Avoiding toxicity, inflammatory responses, or alteration of the function of resident immune cells are important considerations for the development of vaginally applied microbicides. Studies were performed with two classes of candidate microbicide compounds to determine if they would interfere with the recognition of antigen by CD4(+) and CD8(+) T lymphocytes. The presence of nontoxic concentrations of the anionic detergent cholic acid or the sulfated polymer lambda carrageenan did not inhibit recognition of immune peptide by antigen-specific T cells. However, antigen recognition by both CD4(+) and CD8(+) T lymphocytes was inhibited in the presence of the naphthalene sulfonate polymer PRO 2000. Brief (4-h) exposure of antigen-presenting cells or T cells to PRO 2000 did not result in inhibition of antigen uptake and processing by antigen-presenting cells or the ability of specific T cells to respond to antigen stimulation, suggesting that the inhibition was temporary. Binding of antibodies specific for CD18, CD8, and CD3 was impaired in the presence of PRO 2000, suggesting that the mechanism by which this microbicide inhibits T cell recognition of antigenic peptide may involve masking or internalization of surface proteins involved in T cell signaling or stabilizing T cell-antigen-presenting cell interactions. The assays described in this study represent a useful means to screen candidate topical microbicide compounds for inappropriate interactions with immune cells and may be useful for prioritization of candidate microbicide compounds.

Bile Salt/Lecithin Mixed Micelles Optimized for the Solubilization of a Poorly Soluble Steroid Molecule Using Statistical Experimental Design

Bile salts and lecithin combine physiologically to form mixed micelles which aid the solubilization and absorption of dietary fats and drug molecules. In this series of experiments, we have shown how experimental design procedures aid the optimization of a formulation incorporating a bile salt, lecithin, and water with fluticasone propionate (FP) as the model poorly soluble drug. The initial inclusion of a categorical variable ruled out the use of classic response surface designs; therefore the experimental design was constructed using a d-optimal selection from a candidate set of all possible experimental combinations. A separate 2-factor central composite design was used to determine the optimum lecithin and bile salt concentrations over an extended range after the categorical variable had been eliminated. It has been demonstrated that an increase in either lecithin or cholic acid concentration produces an increase in solubility of FP, while sodium taurocholate appears to depress the solubility of FP compared with the other two bile salts. The increase in solubility associated with the increase in bile salt and lecithin is further demonstrated by a linear relationship between FP solubility and the total lipid in the formulation. The influence of molar ratio of lecithin to bile salt in the formulation is also significant. The physical properties of the mixed micellar system (solution turbidity and viscosity ranking) were used to further discriminate between formulations. The optimization showed that the dominant effect was the lecithin, which improves the solubilizing characteristics of the formulation with increasing concentration. The effect of salt concentration is less marked though slightly quadratic in nature. The overall increase in solubility demonstrated was from <1 µg/mL in water to 205 µg/mL in the optimized mixed micellar system.

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 ( P<0.05) along with a 2.9-fold elevation in CA concentration ( P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7α-hydroxylase, 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase and 5β-cholestane-3α,7α,12α-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.