Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM beta-cyclodextrin, 5 mM 2-hydroxypropyl-beta-cyclodextrin, 50 mM SDS and sodium borate-dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C(18)-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 microM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.     

Review: Bacterial transformations of bile acids

Summary The microbial transformation of bile acids that takes place in the lower alimentary tract plays an important role in the in vivo metabolism of bile acids and also of cholesterol in general. Most of the transforming reactions involved can be reproduced in in vitro cultures of mixed intestinal microflora: hydrolysis of the peptide bond in the conjugated bile acids, removal of the 7α-OH group, and dehydrogenation of the α-OH substituents at C-7, C-3 and C-12. The last reaction, which leads to the formation an oxo group, is reversible and a stereospecific reduction of, the oxo moiety into a b-OH group has been shown to be carried out.     

Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation

The content variation dynamics of glucose and free amino acids in blood plasma was followed for lampreys and frogs from autumn till spring, when the exogenous feeding is switched off. In October, the glucose level is rather high (8-10 mM) in blood plasma of both lampreys and frogs. It falls by 50% during winter and falls to a critically low level (1-2 mM) during spring. The lamprey plasma amino acid levels increase by 74% from November to April and thus reach the lower limit known for mammals. The amount of free amino acids in frog plasma decreases by 40% by spring in comparison with the values in autumn. More intensive proteolysis in lamprey tissues compared with that in frog tissues has been confirmed by quantitatively determining leucine, isoleucine, and valine in the blood of these animals. Besides these three amino acids, alanine, glycine, lysine, threonine and, in certain periods, tyrosine have been found to be quantitatively significant in the plasma of both animals.     

ELISA analyses of malate dehydrogenases from nonsulfur purple phototrophic bacteria

Abstract The accumulation of ppGpp in three streptococci starved for isoleucine was studied via HPLC analysis of cell extracts prepared from mechanically disrupted bacteria. Starvation was achieved either by reduction of isoleucine in the growth medium or the addition of pseudomonic acid. The results indicate that while both treatments produced a physiological response similar to that described for stringent strains of other bacteria, in the streptococci, stringency was not necessarily coupled with ppGpp.     

Essential roles of bile acids and their nuclear receptors,FXR and PXR,in the cholestatic liver disease

Bile acids (BAs) are steroid acids found predominantly in the bile of mammals and other vertebrates. Though BAs have been known as digestive juice, recent studies have revealed that BAs act as signaling molecules to control metabolism and inflammation. Today, BAs are considered as potential therapeutic molecules for treatment of complex metabolic liver disease. However, the detergent properties of BAs lead to hepatic injury and intrahepatic cholestasis when BAs are accumulated in the liver with impaired bile flow into gall bladder. Cholestasis is a pathological condition of hepatic retention of cytotoxic bile acids. To date, hydrophilic ursodeoxycholic acid has been currently used to treat cholestasis, but the efficacy of UDCA for cholestasis is still limited. Given that BAs are endogenous ligands of several nuclear receptors, including Farnesoid X receptor and Pregnane X receptor, novel synthetic ligands for those nuclear receptors are promising for the treatment of cholestatic liver diseases.     

Pheromones of the male sea lamprey,Petromyzon marinus L.: structural studies on a new compound, 3-keto allocholic acid,and 3-keto petromyzonol sulfate

This study reports the results of chemical and chromatographic studies which establish the presence of 3-keto allocholic acid (3kACA) in water extracts from spermiating male sea lamprey, Petromyzon marinus. This is the second compound to be isolated and identified from these extracts. The first was 3-keto petromyzonol sulfate (3kPZS), which was shown to act as strong pheromonal attractant for ovulated females. Some new characterization data on 3kPZS (utilizing an only recently available synthetic preparation of the compound) is also included. The possibility that a mixture of 3kACA and 3kPZS might be a more potent pheromonal attractant than either compound alone is discussed.     

Synthesis and haemolytic activity of novel salts made of nicotine alkaloids and bile acids

A series of novel salts made of nicotine alkaloids and bile acids were synthesized and their haemolytic activity was examined in vitro using human erythrocytes. All compounds were characterized by spectroscopic methods. The novel salts show membrane-perturbing properties inducing the erythrocyte shape alterations and haemolysis in dose-dependent manner. Nicotine decreases the membrane interacting potential of bile acids in the novel compounds. The presence of sulfur or selenium atom in the nicotine molecule affects the haemolytic activity of its novel salts depending on the hydrophobicity of bile acids.     

Synthesis and antimicrobial evaluation of bile acid tridentate conjugates

Two series of novel bile acid tridentate conjugates with different linkers were synthesized and characterized, and their biological activities in vitro were evaluated. The procedure was straightforward and efficient to be carried out with high overall yield. The antimicrobial activity of the synthesized compounds against Saccharomyces cerevisiae, Aspergillus niger, Escherichia coli and Staphylococcus aureus was investigated in vitro. The best activity of minimal inhibitory concentrations (MICs) for 1c, 1c′, 2c and 2c′ against S. cerevisiae was up to 0.125 μg/mL.     

Synthesis of 24-phenyl-24-oxo steroids derived from bile acids by palladium-catalyzed cross coupling with phenylboronic acid. NMR characterization and X-ray structures

Palladium-catalyzed cross coupling of phenyboronic acid with acetylated bile acids in which the carboxyl functions have been activated by formation of a mixed anhydride with pivalic anhydride afforded moderate to good yield of 24-phenyl-24-oxo-steroids. Unambiguous assignments of the NMR signals were made with the aid of combined 1D and 2D NMR techniques. X-ray diffraction studies confirmed the obtained structures.     

Capillary gas chromatographic analysis of serum bile acids as the n-butyl ester–trimethylsilyl ether derivatives

Gas chromatographic separations of n-butyl ester-trimethylsilyl ether derivatives of several common bile acids were compared with those of the corresponding methyl ester-trimethylsilyl ether derivatives on a CP-Sil-5 CB capillary column. Both types of derivatives were similarly resolved from each other. However, the n-butyl ester-trimethylsilyl ether derivatives of the bile acids showed longer retention times than the corresponding methyl ester-trimethylsilyl ethers and unlike the methyl ester-trimethylsilyl ether derivatives, were completely resolved from and eluted later than the trimethylsilyl ethers of common plasma sterols including sitosterol. A simplified method of plasma work-up for quantitation of bile acids and application of the above method in quantification of plasma bile acids in humans is described.     

New highly toxic bile acids derived from deoxycholic acid,chenodeoxycholic acid and lithocholic acid

We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features individually associated with cytotoxicity in our previous work. The effect of the compounds on cell viability of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further studied using flow cytometry in order to understand the mechanism of cell death. Several compounds were identified with low micromolar IC₅₀ values for reducing cell viability in the Caco-2 and HT1080 cell lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death induction by BAs may be structure–specific. Compounds derived from DCA caused cell death through apoptosis. There was some evidence of selectivity between the two cell lines studied which may be due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects. The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced cell death. Compound 8 could be useful for investigating this phenomenon.     

Quantitation of free and conjugated bile acids in human feces using a high-pressure liquid chromatography counterion method

A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method.     

Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC

An ELISA Microtiter Plate, Ochratoxin Test called AgraQuant^® was validated to measure ochratoxin A in a range from 2 to 40ppb in corn, milo, barley, wheat, soybeans and green coffee. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, ochratoxin A is extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 10min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450nm. Results obtained from internal validation studies assessing accelerated stability indicate a 1year shelf life; accuracy and precision are comparable to HPLC from 0 to 80ppb and limit of detection in corn is 1.9 ppb and other food commodities is up to 3.8 ppb. Comparison of the method to HPLC, ability to detect individual ochratoxins, and ruggedness of the test kits determined this test to be rugged from 18 to 30°C, sensitive, accurate, precise and effective comparable to HPLC for measuring ochratoxin A ranging from 2 to 40ppb in several commodities.     

First synthesis of phosphonobile acids and preliminary studies on their aggregation properties

The synthesis of three novel phosphonobile acids from natural bile acids is reported. The CMC of phosphonodeoxycholic acid (PDCA) at pH 8.2 was found to be lower than that of the parent deoxycholic acid (DCA). PDCA micelles were also found to have higher microviscosity compared to DCA micelles, suggesting higher hydrophobicity and tighter packing in the interior of PDCA micelles. PDCA aggregated further to form an aqueous gel at pH 4.