deoxycholic-acid and Hemolysis

This report describes a case of hemolysis in a patient injecting deoxycholic acid and benzyl alcohol for aesthetic benefit without medical supervision. The concentration and dose injected by the patient resulted in a 10-fold overdose of deoxycholic acid in comparison to the FDA-recommended dosing for the approved indication. Providers should be aware of medically unsupervised use of DCA and other injectables and the potential risks associated with this practice.

Topics: Adult; Cholagogues and Choleretics; Deoxycholic Acid; Hemolysis; Humans; Injections, Subcutaneous; Male

Colorectal cancer (CRC) is a prevalent and fatal cancer. Oral administration provided the potential for in situ treatment of the colorectal cancer. However, drugs couldn't be well-absorbed mainly due to its degradation in the gastric area and poor intestinal permeability. In this study, we synthesized deoxycholic acid and hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) as oral curcumin (CUR) delivery system for colorectal cancer treatment. DAHBC with lower critical solution temperature (LCST) below 37 °C (27-33 °C) was obtained. DAHBC NPs were correspondingly stable in simulated gastric conditions (pH 1.2, 37 °C), due to the offset of size change between pH-responsive expansion and thermo-responsive shrinkage. In simulated intestinal tract (pH 7.0-7.4, 37 °C), DAHBC NPs exhibited burst release of CUR owing to the onefold effect of thermo-responsive shrinkage. DAHBC27 NPs showed the minimum CUR leakage (~10%) in simulated gastric conditions, because a furthest temperature-sensitive shrinkage caused by the lowest LCST offset the expansion in acid environment. DAHBC27 NPs induced ~10-fold increased (P < 0.05) CUR absorption by paracellular transport pathway, compared to the free CUR. Thus, DAHBC NPs stabilized in the gastric environment may be a promising oral drugs delivery system for effective in situ colorectal cancer therapy.

Topics: Administration, Oral; Adsorption; Caco-2 Cells; Chitosan; Colorectal Neoplasms; Curcumin; Deoxycholic Acid; Drug Carriers; Drug Liberation; Drug Stability; Gastric Mucosa; Hemolysis; Humans; Intestinal Absorption; Materials Testing; Nanoparticles; Temperature

Amphotericin B (AmB) is widely applied in treatment of systemic fungal infections. However, the emergence of severe adverse effects, such as nephrotoxicity, hepatotoxicity and hemolytic anemia, can limit its clinical use. Poly(lactide-co-glycolide) (PLGA) or poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) blend nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. Nanoparticles were characterized by particle size, polydispersity index, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction analyses. The antifungal activity was evaluated against strains of Candida albicans and Cryptococcus neoformans. Toxicity was evaluated by hemolysis assay and after 7 days treatment in rats. Mean nanoparticle size was below 200nm with low polydispersity and AmB encapsulation efficiency higher than 90%. Nanoencapsulation resulted in AmB amorphization and no chemical interaction between drug and polymer. In C. albicans, minimum inhibitory concentration (MIC) of AmB-loaded PLGA-PEG nanoparticles was 2-fold higher than free AmB or marketed deoxycholate AmB (AmB-D), while MIC of AmB-loaded PLGA nanoparticles was 10-fold higher than AmB-loaded PLGA-PEG. In C. neoformans, the efficacy of AmB-loaded PLGA nanoparticles was comparable to free AmB, while AmB-loaded PLGA-PEG nanoparticles and AmB-D did not present MIC in tested concentration range. Nanoparticles inhibited the AmB-induced hemolysis. After 7 days treatment in rats, histologic examination revealed AmB-D treatment presented initial liver damage, while AmB-loaded nanoparticles did not present any hepatic cellular alteration. Kidney alteration was not observed in all treatments. Thus, PLGA and PLGA-PEG nanoparticles are promising carriers for AmB delivery with potential application in antifungal therapy.

Topics: Amphotericin B; Animals; Antifungal Agents; Candida albicans; Cryptococcus neoformans; Deoxycholic Acid; Drug Combinations; Drug Delivery Systems; Hemolysis; Humans; Male; Microbial Sensitivity Tests; Nanoparticles; Particle Size; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Wistar; Toxicity Tests

This study presents the mode of interaction, structural features, and micellization of amphotericin B (AmB) with sodium deoxycholate sulfate (SDCS) as small lipid molecule at different ratios, as revealed by molecular docking simulations and nuclear magnetic resonance (NMR). AmB-SDCS micelles were synthesized by single pot rinsing method. Solid-state

Topics: Amphotericin B; Chemistry, Pharmaceutical; Deoxycholic Acid; Drug Carriers; Erythrocytes; Freeze Drying; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Lipids; Magnetic Resonance Spectroscopy; Micelles; Molecular Docking Simulation; Nanoparticles; Particle Size

Effective treatments for tumors are not easy to achieve due to the existence of metastases, which are responsible for most tumor death. Hence, a new drug delivery system is a pressing need, which should be biocompatible, stimuli-responsive, and multifunctional, including antitumor, antimetastasis, and antiangiogenesis effects. However, it is challenging to achieve all of these properties in one drug delivery system. Here, we developed a system of drug DOX and heparin into one self-assemble nanoparticle via pH-sensitive hydrazone bond and hydrophobic groups, deoxycholate. In the process, heparin itself was not only as the hydrophilic segments of the carrier, but also processed multiple biological functions such as antiangiogenesis and antimetastasis effect. The micelle nanoparticle HD-DOX processed good stability and acidic pH-triggered drug release property. After systemic administration, heparin-based micelle nanoparticle showed longer half-time and enhanced accumulation of DOX in tumors through the enhanced permeability and retention effect, leading to more efficient antitumor effects. In addition, heparin could hinder platelet-induced tumor cells epithelial-mesenchymal transition (EMT) and partially affect cell actin cytoskeletal arrangement, resulting in the disorganization of the actin cytoskeleton. Therefore, HD-DOX exhibited significant inhibitory effect on the metastasis in melanoma animal model in C57BL/6 mouse. Meanwhile, benefited from the antiangiogenesis effect of heparin, tube formations in endothelial cells were effectively inhibited and tumor vascular density was decreased by HD-DOX. Taken together, our study developed a self-assembly nanoplatform that both the drug and carrier had therapeutic effects with ideal antitumor efficacy.

Topics: Actin Cytoskeleton; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Blood Platelets; Cell Death; Cell Line, Tumor; Cell Movement; Deoxycholic Acid; Doxorubicin; Drug Carriers; Erythrocytes; Hemolysis; Heparin; Human Umbilical Vein Endothelial Cells; Humans; Hydrodynamics; Injections, Intravenous; Mice, Inbred C57BL; Micelles; Nanoparticles; Neoplasm Metastasis; Neovascularization, Physiologic; Tissue Distribution

A bio-inspired nanocarrier was developed for protein delivery based on biodegradable amphiphilic chitosan derivative (DCA-PCCs) with hydrophilic cell membrane mimic phosphorylcholine (PC) and hydrophobic deoxycholic acid (DCA) moieties, which was synthesized via the combination of Atherton-Todd reaction and carbodiimide coupling reaction. Using bovine serum albumin (BSA) as model protein, it was found that DCA-PCCs with suitable degree of substitution of PC and DCA moieties can load proteins by forming nanocomplexes via a solvent evaporation method. The physicochemical characteristics of BSA/DCA-PCCs nanocomplexes were investigated by Zetasizer, atomic force microscopy (AFM) and Fourier-transform infrared (FT-IR) spectroscopy. In vitro biological evaluation revealed BSA/DCA-PCCs nanocomplexes as blank DCA-PCCs nanoparticles had excellent cytocompatibility and hemocompatibility mainly due to the presence of cell membrane mimic phosphorylcholine. BSA release results suggested BSA/DCA-PCCs nanocomplexes showed a sustained release behavior following first order exponential decay kinetics. The results indicated DCA-PCCs provided a promising approach for effectively delivering therapeutic proteins.

Topics: Animals; Biomimetic Materials; Cattle; Chitosan; Deoxycholic Acid; Drug Carriers; Drug Liberation; Hemolysis; Hydrophobic and Hydrophilic Interactions; Mice; Nanoparticles; NIH 3T3 Cells; Phosphorylcholine; Rabbits; Serum Albumin, Bovine; Solvents

Novel biomimetic amphiphilic chitosan derivative, deoxycholic acid-phosphorylcholine-chitosan conjugate (DCA-PCCs) was synthesized based on the combination of Atherton-Todd reaction for coupling phosphorylcholine (PC) and carbodiimide coupling reaction for linking deoxycholic acid (DCA) to chitosan. The chemical structure of DCA-PCCs was characterized by (1)H and (31)P nuclear magnetic resonance (NMR). The self-assembly of DCA-PCCs in water was analyzed by fluorescence measurements, dynamic laser light-scattering (DLS), zeta potential and transmission electron microscopy (TEM) technologies. The results confirmed that the amphiphilic DCA-PCCs can self-assemble to form nanosized spherical micelles with biomimetic PC shell. In vitro biological evaluation revealed that DCA-PCCs micelles had low toxicity against NIH/3T3 mouse embryonic fibroblasts as well as good hemocompatibility. Using quercetin as a hydrophobic model drug, drug loading and release study suggested that biomimetic DCA-PCCs micelles could be used as a promising nanocarrier avoiding unfavorable biological response for hydrophobic drug delivery applications.

Topics: Animals; Biomimetic Materials; Cell Survival; Chitosan; Deoxycholic Acid; Drug Carriers; Erythrocytes; Hemolysis; Mice; Micelles; NIH 3T3 Cells; Particle Size; Phosphorylcholine; Polymers; Quercetin; Rabbits; Spectrometry, Fluorescence

The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri , providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is composed of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g., deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. Although the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study because of the hydrophobic nature of the IpaB and IpaC translocator proteins. Here, we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11-27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together, these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation.

Topics: Amino Acid Substitution; Antigens, Bacterial; Bacterial Proteins; Bacterial Secretion Systems; Deoxycholic Acid; Fluorescence Resonance Energy Transfer; Hemolysis; Models, Molecular; Mutagenesis, Site-Directed; Peptide Fragments; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Shigella flexneri; Virulence Factors

The aim of this work was to evaluate how an aqueous micellar system containing Amphotericin B (AmB) and sodium deoxycholate (DOC) can be rebuilt after heating treatment. Also, a review of the literature on the physicochemical and biological properties of this new system was conducted. Heated (AmB-DOC-H) and unheated (AmB-DOC) micelles were then diluted at four different concentrations (50 mg · L(-1), 5 mg · L(-1), 0.5 mg · L(-1), and 0.05 mg · L(-1)) to perform physicochemical studies and a pharmacotoxicity assay, in which two cell models were used for the in vitro experiments: red blood cells (RBC) from human donors and Candida parapsilosis (Cp). While potassium (K(+)) and hemoglobin leakage from RBC were the parameters used to evaluate acute and chronic toxicity, respectively, the efficacy of AmB-DOC and AmB-DOC-H were assessed by K(+) leakage and cell survival rate from Cp. The spectral study revealed a slight change in the AmB-DOC aggregate peak from 327 nm to 323 nm, which is the peak for AmB-DOC-H. Although AmB-DOC and AmB-DOC-H exhibited different behavior for hemoglobin leakage, AmB-DOC produced higher leakage than AmB-DOC-H at high concentrations (from 5 mg · L(-1)). For K(+) leakage, both AmB-DOC and AmB-DOC-H showed a similar profile for both cell models, RBC and Cp (P < 0.05). AmB-DOC-H and AmB-DOC also revealed a similar profile of activity against Cp with an equivalent survival rate. In short, AmB-DOC-H showed much less toxicity than AmB-DOC, but remained as active as AmB-DOC against fungal cells. The results highlight the importance of this new procedure as a simple, inexpensive, and safe way to produce a new kind of micelle system for the treatment of systemic fungal infections.

Topics: Amphotericin B; Analysis of Variance; Candida; Deoxycholic Acid; Dose-Response Relationship, Drug; Erythrocytes; Hemoglobins; Hemolysis; Hot Temperature; Humans; Micelles; Microbial Viability; Models, Biological; Nanotechnology; Potassium; Spectrophotometry, Ultraviolet

The objective of this work was to study the effect of structure of bile acids on their membranolytic potential and extent of overlapping of the information about the membranolytic potential of bile acids and their physico-chemical parameters, namely: retention index R(M0) (as a measure of bile acid hydrophobicity, reversed-phase thin-layer chromatography (RPTLC)), lecithin solubilisation (measure of the interaction of bile acids with phospholipids) and critical micellar concentration (CMC). It was found that bile acid concentrations at 100% lysis of erythrocyte membranes is described best by their CMC values, whereas at 50% lysis the parameter used is lecithin solubilisation. This indicates that different mixed micelles are formed in the membrane lysis at lower and higher concentrations of bile acids. Replacement of the hydroxyl (OH) group in the bile acid molecule with an oxo group yields derivatives with lowered hydrophobicity, power of lecithin solubilisation, tendency for self-aggregation as well as the membranolytic activity.

Topics: Animals; Chenodeoxycholic Acid; Cholagogues and Choleretics; Cholic Acid; Deoxycholic Acid; Erythrocytes; Gastrointestinal Agents; Hemolysis; Hydrophobic and Hydrophilic Interactions; Lecithins; Models, Molecular; Molecular Structure; Rabbits

Bufadienolides-loaded nanostructured lipid carriers (BU-NLC) were prepared for parenteral application using glyceryl monostearate as solid core, medium-chain triglyceride and oleic acid as liquid lipid material, and Lipoid E-80, sodium deoxycholate and pluronic F68 as stabilizers. In this study, the in vitro cytotoxicity, pharmacokinetics, biodistribution, antitumor efficacy and safety of BU-NLC were evaluated. Against human astrocytoma cell line (U87-MG) and human gastric carcinoma cell line (HGC-27) BU-NLC exhibited cytotoxicity that was similar to that of the free drug, and superior to that of the commercially available fluorouracil injection. BU-NLC exhibited a linear pharmacokinetic behavior at doses ranging from 0.25 to 1.0 mg/kg. The improved pharmacokinetic profile of bufadienolides when formulated in BU-NLC resulted in a higher plasma concentration and lower clearance after intravenous administration compared with bufadienolides solution (BU-S). A biodistribution study indicated that bufadienolides were mainly distributed in the lung, spleen, brain and kidney, and the longest retention was observed in the brain. A sarcoma-180 tumor model further confirmed the advantages of BU-NLC versus BU-S. Hemolysis and acute toxicity investigations showed that BU-NLC was safe when given by intravenous injection with reduced toxicity. In conclusion, the NLC system is a promising approach for the intravenous delivery of bufadienolides.

Topics: Animals; Antineoplastic Agents; Bufanolides; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Deoxycholic Acid; Dose-Response Relationship, Drug; Drug Carriers; Drug Compounding; Excipients; Female; Glycerides; Hemolysis; Humans; Inhibitory Concentration 50; Injections, Intravenous; Lethal Dose 50; Lipids; Male; Mice; Nanostructures; Oleic Acid; Poloxamer; Rabbits; Rats; Rats, Wistar; Sarcoma 180; Technology, Pharmaceutical; Tissue Distribution; Triglycerides

The mechanism which protects the biliary and intestinal mucosa from the detergent properties of bile acids is not fully understood. We employed three contrasting in vitro model systems (human red blood cells, polarized intestinal [Caco-2] cells, and synthetic liposomes), to compare the efficacy of saturated and unsaturated phosphatidylcholine (PC) to protect cells and membranes from bile salt injury.. Hemolysis of red blood cells, electrical resistance across confluent monolayers of Caco-2 cells, and disruption of synthetic PC liposomes were assessed after incubation with varying concentrations of bile salt (sodium deoxycholate) alone or in the presence of saturated or unsaturated PC.. The hemolytic activity of deoxycholate on red blood cells was observed at > or =2 mM, and could be blocked by equimolar concentration or greater of both saturated or unsaturated PC. In contrast, exposure of Caco-2 cells to deoxycholate at > or =0.8 mM induced a maximal decrease in resistance, which was reversed by > or =0.8 mM unsaturated PC or 5 mM saturated PC. Similarly, synthetic liposomes were permeabilized by 0.8 mM deoxycholate and were protected by a lower concentration of unsaturated PC (2 mM) than saturated (5 mM).. Cells can show variable resistance to bile salt toxicity. Extracellular PC, especially in the unsaturated state, can directly protect cell and artificial membranes from bile salt injury. These findings support a role for biliary PC in the formation of mixed micelles that have low cytotoxic properties.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Caco-2 Cells; Cell Membrane; Cell Membrane Permeability; Cytoprotection; Deoxycholic Acid; Dose-Response Relationship, Drug; Electric Impedance; Epithelial Cells; Erythrocytes; Hemolysis; Humans; Intestinal Mucosa; Liposomes; Phosphatidylcholines; Protective Agents; Time Factors

In the present study, several nasal absorption enhancers, used in metoclopramide hydrochloride (MCP HCl) nasal solutions, have been screened for their possible damaging effect in the in vitro human erythrocytes lysis experiment. Moreover, the in vivo leaching of biological markers from the rat nasal epithelium was used as a quantitative assessment for possible nasal mucosal irritation whereby the extent of release of total protein and lactate dehydrogenase (LDH) in the nasal lavage fluid was determined. Results showed that insignificant hemolysis from normal saline (P<0.05) occurred with the enhancer protamine sulphate while poly-l-arginine and sodium cholate demonstrated very low (<15%) hemolysis and caused insignificant protein and LDH release from the rat nasal mucosa. Conversely, sodium deoxycholate and chitosan polymers (either of low or high molecular weight) showed high (>60%) hemolysis in vitro and the release of the biological markers in vivo was significantly higher (P<0.05) than the control solution (no enhancer). A significant correlation (P<0.05) existed between the enhancement effect of MCP HCl nasal absorption and the amounts of protein (r=0.85) and LDH (r=0.88). Furthermore, the pharmacokinetics of MCP HCl was determined after intravenous (IV), per-oral and intranasal administration of 10mg drug dose in rabbits. The application of a nasal spray (NS) solution containing 0.5% sodium cholate resulted in a significant improvement (P<0.05) in both the rate and extent of absorption of MCP HCl where the T(max) achieved was 23.3min as compared to 50min in case of the oral solution while the area under the serum concentration-time curve (AUC(0-infinity)) were 506.1, 434.9 and 278.7microg/mlmin for IV, NS and oral solutions, respectively. These values corresponded to absolute bioavailabilities of 87.21 and 55.61% for the NS and oral solutions, respectively. It could thus be concluded that NS of MCP HCl represents a viable approach to achieving rapid and high systemic drug absorption during the emergency treatment of severe emesis.

Topics: Absorption; Administration, Intranasal; Animals; Antiemetics; Biological Availability; Chitosan; Deoxycholic Acid; Erythrocytes; Hemolysis; Humans; L-Lactate Dehydrogenase; Male; Metoclopramide; Nasal Lavage Fluid; Nasal Mucosa; Peptides; Protamines; Proteins; Rats; Rats, Inbred Strains; Sodium Cholate

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.

Topics: Alanine Transaminase; Aspartate Aminotransferases; Deoxycholic Acid; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Humans; Taurochenodeoxycholic Acid; Taurodeoxycholic Acid; Time Factors; Transglutaminases; Tumor Cells, Cultured

The survival of Lactobacillus reuteri when challenged with glycodeoxycholic acid (GDCA), deoxycholic acid (DCA) and soygerm powder was investigated. Moreover, the impact of Lact. reuteri on the bioavailability of isoflavones present in soygerm powder was examined.. The strain experienced a die-off when adding 2 or 3 mmol l-1 bile salts, with more pronounced effects in the case of DCA. By means of a haemolysis test it was shown that toxicity could be due to membrane damage. When 4 g l-1 soygerm powder was added, the Lactobacillus strain survived the bile salt burden better (P < or = 0.05) and the membrane damage in the haemolysis test decreased (P < or = 0.05). The Lact. strain cleaved beta-glycosidic isoflavones during fermentation of milk supplemented with soygerm powder.. The interactions between the Lactobacillus strain and soygerm powder suggest that combining both in fermented milk can exhibit advantageous probiotic effects. The relevance of the combination of the strain and the soygerm powder should be studied under more relevant physiological conditions.

Topics: Animals; Deoxycholic Acid; Detergents; Fermentation; Glycine max; Glycodeoxycholic Acid; Hemolysis; Isoflavones; Lactobacillus; Milk; Probiotics

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.

Topics: Bile Acids and Salts; Cell Membrane Permeability; Deoxycholic Acid; Detergents; Erythrocyte Membrane; Glycodeoxycholic Acid; Hemolysis; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Taurodeoxycholic Acid

The marine natural product, halistanol trisulfate, has a relatively low critical micelle concentration of 0.001% m/v (14.5 microM) and strong hemolytic potency with an EC50 of 0.00046% m/v (6.67 microM). As expected of a detergent, it inhibits the growth of gram-positive but not gram-negative bacteria. The hemolytic activity of halistanol trisulfate and other detergents has been shown to correlate with critical micelle concentration. This correlation may have important implications in the mechanism of membranolytic bioactivity.

Topics: Animals; Bacillus cereus; Deoxycholic Acid; Detergents; Escherichia coli; Hemolysis; Humans; Micelles; Microbial Sensitivity Tests; Octoxynol; Polyethylene Glycols; Porifera; Pseudomonas aeruginosa; Staphylococcus aureus; Sterols

The membrane damaging potential of dilute solutions of bile salts was evaluated by monitoring continuously the hemolysis of a small sample of red blood cells (RBC) introduced into a defined media containing the bile salts at various pH values. The strength of the hemolytic bile salt was characterized by the rate of the induced hemolysis and by the time that elapsed between the introduction of the RBC sample into the bile salt containing solution and the onset of hemolysis. The potency of the unconjugated bile acids was extremely sensitive to pH, e.g. the rate of hemolysis caused by a 7.5 mM cholate was 1.5%, 20% and 64% per min when the pH of the solution was 7.65, 7.3 and 6.85, respectively. At low pH values the membrane damaging effects of deoxycholate was clearly discerned at micromolar concentration range. The hemolytic potency of glycodeoxycholate was also enhanced significantly by lowering the pH. The taurine-conjugated cholate and deoxycholate were only slightly sensitive to variations in pH. Taurocholate at concentrations that were not hemolytic greatly enhanced the injurious potency of deoxycholate. These results imply that in acidic solutions the presence of bile acids can cause damage to cell membranes. It is suggested that the acidic environment in the proximal duodenum and acidosis developed during hypoxia in the liver are two situations in which the bile salts may constitute a pathogenic factor.

Topics: Animals; Bile Acids and Salts; Chenodeoxycholic Acid; Cholic Acid; Cholic Acids; Deoxycholic Acid; Erythrocytes; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Rats; Structure-Activity Relationship; Ursodeoxycholic Acid

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.

Topics: Aminoquinolines; Bile Acids and Salts; Calcium; Calcium Chloride; Deoxycholic Acid; Dose-Response Relationship, Drug; Edetic Acid; Hemolysis; Humans; In Vitro Techniques; Potassium Chloride; Sodium Chloride; Time Factors

The binding of C8 and C9 from human serum to target erythrocytes was quantified, and the molecular stoichiometries of C9:C8 within terminal C5b-9(m) complexes were determined. Low doses of serum generated terminal complexes with mean C9:C8 ratios of 2 to 3:1, whereas complexes generated by highest serum doses harbored an average of six to eight C9/C8 molecules. From the collective biochemical and ultrastructural data, we concluded that heterogeneous populations of terminal complexes regularly form on target membranes; those containing high numbers of C9 molecules (greater than or equal to six to eight) exhibit the structure of the classical "lesion", whereas those containing low numbers of C9 do not exhibit this typical structure, although they probably still function as small pores. A major cause for this heterogeneity of the lesions derives from shortage of C9, which is naturally present in a 2 to 1 molar ratio relative to C8 in serum. Generation of terminal complexes harboring high numbers of C9 on erythrocyte membranes is possible in spite of this natural shortage because SC5b-9 does not form in the fluid phase to compete for C9 binding. If interrupted, the process of C9-C9 oligomerization cannot be recontinued, and "incomplete" C5b-9 complexes are unable to bind additional C9 upon reincubation with this component. The demonstrated heterogeneity of terminal complexes with respect to their C9 content may explain the functional heterogeneity of complement lesions observed previously by other investigators.

Topics: Animals; Blood Physiological Phenomena; Centrifugation, Density Gradient; Complement C8; Complement C9; Complement Membrane Attack Complex; Complement System Proteins; Deoxycholic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Rabbits; Receptors, Complement

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.

Topics: Animals; Cattle; Deoxycholic Acid; Deuterium; Erythrocyte Membrane; Hemolysis; Humans; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphorus; Swine

Topics: Deoxycholic Acid; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Methods; Saponins; Sodium; Sodium-Potassium-Exchanging ATPase

A culture product of Streptomyces pseudovenezuelae MF722-02, with a molecular formula of C29H32N2O7, was isolated as yellow needles from culture broths and mycelia of the organism by means of a series of solvent extraction, column chromatography and crystallization. The antibiotic is active against some Gram-positive bacteria, inhibits growth in vitro of cells of mouse leukemia L-1210, prolongs the life span of mice inoculated with the leukemia cells, enhances deoxycholate-induced hemolysis in vitro and inhibits (Na+, K+)-ATPase in vitro.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Bacteria; Cell Membrane; Deoxycholic Acid; Hemolysis; Leukemia L1210; Mice; Micrococcus; Sodium-Potassium-Exchanging ATPase; Streptomyces

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte 'ghosts' was solubilized and reconstituted to form membranous vesicles capable of energized Ca2+ accumulation. The erythrocyte 'ghosts' for this purpose were prepared by using isoosmotic freeze-haemolysis in the presence of Tween 20 and proteinase inhibitors to stabilize the preparation. The reconstitution procedure is similar to that developed by Meissner & Fleischer [(1974) J. Biol. Chem. 249, 302-309] for skeletal-muscle sarcoplasmic-reticulum in that: (1) deoxycholate is used for the solubilization of the membrane; (2) controlled dialysis at near room temperature, rather than 0 degree C, is required in order to obtain a functional preparation capable of Ca2+ accumulation; and (3) membrane vesicles can be reassembled with protein/lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original membrane.

Topics: Biological Transport; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Deoxycholic Acid; Dialysis; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Methods; Polysorbates

Topics: Bile Acids and Salts; Cholic Acids; Deoxycholic Acid; Erythrocytes; Glycocholic Acid; Hemolysis; Humans; Phospholipids; Polyethylene Glycols; Taurocholic Acid

1. Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography. 2. The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine. This preparation was free from protease, lipase and oxygen-labile delta-hemolysin. 3. Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates. 4. Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme. 5. This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine.

Topics: Animals; Calcium; Clostridium; Deoxycholic Acid; Erythrocytes; Hemolysis; Horses; Hot Temperature; Hydrogen-Ion Concentration; Magnesium; Molecular Weight; Phosphatidylcholines; Phospholipases; Phospholipids; Surface-Active Agents

The effect of several surface-active substances (Na-oleate, Na-desoxycholate, Triton X 100, Pluronic F 38, Pluronic F 68, Pluronic F 108) on dextran-induced red cell aggregation (RCA) and red cell shape was investigated. Sedimentation measurements and photometric determinations of RCA indicated a desaggregating effect of oleate, Triton, F 38 and F 68. Shape changes were found in the presence of oleate and desoxycholate (crenation) and Triton (cup formation); in addition, these substances induced pronounced hemolysis. No hemolysis and no shape changes were found with any of the Pluronic polyols.

Topics: Blood Sedimentation; Cell Aggregation; Deoxycholic Acid; Depression, Chemical; Dextrans; Erythrocytes; Hemoglobins; Hemolysis; Humans; In Vitro Techniques; Oleic Acids; Polyethylene Glycols; Polyethylenes; Sodium; Stimulation, Chemical; Surface-Active Agents

In a previous paper we have discussed Shihabi and Bishop's method for the determination of lipase activity and proposed some modifications. The results of further studies are reported. An initial rise in absorbance which we found with some sera appeared to be caused by an interaction between desoxycholate and lipoproteins; this effect could be removed by treating the sera with dextrane sulphate prior to the determination of the enzyme activity. In urine no lipase activity can be demonstrated; urine contains no inhibitory substances. Red blood cells do not contain lipase and even fairly intense haemolysis does not interfere with our method. Lipase and amylase activities in patients' sera are compared. A good correlation between our method and that of Rick was found.

Topics: Amylases; Deoxycholic Acid; Evaluation Studies as Topic; Hemolysis; Humans; Kinetics; Lipase; Methods; Time Factors

Topics: Animals; Bacillus cereus; Cell Membrane; Chromatography, Thin Layer; Deoxycholic Acid; Erythrocytes; Hemolysis; Humans; Indicators and Reagents; Lipids; Methods; Pancreas; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipases; Phospholipids; Polyethylene Glycols; Sphingomyelins; Swine

Topics: Adenosine Triphosphatases; Bile Acids and Salts; Cell Membrane; Cold Temperature; Colloids; Deoxycholic Acid; Drug Stability; Edetic Acid; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Macromolecular Substances; Ouabain; Potassium; Sodium; Spectrophotometry, Ultraviolet; Surface Tension; Time Factors

Topics: Cations, Monovalent; Cell Membrane; Cesium; Deoxycholic Acid; Detergents; Drug Synergism; Erythrocytes; Hemolysis; Humans; Lithium; Potassium; Rubidium; Sodium

Topics: Animals; Antigens, Viral; Centrifugation, Density Gradient; Centrifugation, Zonal; Chick Embryo; Complement Fixation Tests; Culture Techniques; Deoxycholic Acid; Encephalitis Viruses; Encephalomyelitis, Equine; Ethyl Ethers; Hemagglutination Tests; Hemolysis; Immune Sera; Rabbits; Sodium Dodecyl Sulfate; Surface-Active Agents; Viral Plaque Assay; Viral Proteins; Virus Cultivation

Topics: Alkylation; Animals; Antigen-Antibody Reactions; Antigens; Binding Sites, Antibody; Blood Proteins; Cell Membrane; Cholesterol; Chromatography, Gel; Deoxycholic Acid; Epitopes; Erythrocytes; Hemolysis; Lipids; Mercaptoethanol; Oxidation-Reduction; Phospholipids; Potassium; Sheep; Sodium

Topics: Albumins; Alcaligenes; Calcium; Chromatography; Chromatography, Gel; Deoxycholic Acid; Edetic Acid; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Magnesium; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases; Protamines; Sphingomyelins; Temperature; Ultrafiltration; Zinc