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PubMed1994

Comparison of micellar electrokinetic chromatography (MEKC) with capillary gas chromatography in the separation of phenols, anilines and polynuclear aromatics potential field-screening applications of MEKC.

Capillary electrophoresis (CE) is known to be complementary to liquid chromatography, but comparison of CE with capillary gas chromatography (GC) for applicable analytes has not been extensive. Capillary GC has been the preeminent separation technique for environmental analysis, but CE has yet to be applied systematically to the determination of environmental analytes. We present data on separations of three classes of semivolatile analytes of interest to environmental analysis: phenols, anilines and polynuclear aromatic hydrocarbons (PNAs). Standard GC conditions were used to illustrate typical separations observed on 30-m and 40-m columns. Rapid analyses were addressed using a high-temperature 15-m column of thinner film. CE separations employed borate buffer with sodium cholate as the micellar agent in micellar electrokinetic chromatography (MEKC). The effects of organic additives were studied using methanol, acetone and tetrahydrofuran. gamma-Cyclodextrin was also used in MEKC to enhance the separation of polynuclear aromatic hydrocarbons and to examine its effects on separations of phenols and anilines. Short capillaries effected very rapid (< 3 min) compound-class characterization, an approach which has potential use in site characterization/remediation (field-screening) studies.

Aniline Compounds↗

PubMed1994

High-performance capillary electrophoresis for characterization of hapten-protein conjugates used for production of antibodies against soyasaponin I.

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten-protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-alpha-D-galactoside; PNPG) coupled to KSTI. The PNPG-KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten-protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

Animals↗

PubMed1996

Quantitative structure-activity relationships studies with micellar electrokinetic chromatography. Influence of surfactant type and mixed micelles on estimation of hydrophobicity and bioavailability.

Applications of micellar electrokinetic chromatography (MEKC) in quantitative structure-activity relationships (QSAR) were studied. First, quantitative structure-retention relationships (QSRR), which describe the correlation between logarithm of capacity factor (log k') in MEKC and logarithm of distribution coefficient between 1-octanol and water (log P(ow)), were investigated for 60 aromatic compounds and 9 corticosteroids using three different anionic surfactants [e.g., sodium dodecyl sulfate (SDS), sodium cholate (SC), and lithium perfluorooctane sulfonate (LiPFOS)], one cationic surfactant (C14TAB), and mixed anionic micellar systems. Linear solvation energy relationships (LSER) and solvatochromic parameters were used to shed light on the different log k' vs. log P(ow) relationships of the various surfactants. It was concluded that hydrogen bonding interactions have a great influence on retention behavior in MEKC and its relationships with hydrophobicity. Interestingly, bile salt surfactants (e.g., SC) and mixed bile salt micellar systems provide better correlations for log k' vs. log P(ow) than SDS and/or SDS with buffer additives (e.g., beta-cyclodextrin, urea, and acetonitrile). Using SC micelles, only one line was adequate to describe the relationship between retention in MEKC and hydrophobicity for a group of 60 aromatic compounds. The existence of higher correlation for the SC system was attributed to a similar hydrogen bonding pattern between SC micelles and 1-octanol. In the SDS system, however, three lines were recognized for the congeneric subgroups of compounds. This is due to the hydrogen bond door (HBD) characteristic of SDS micelles that selectively differentiate between the solutes with different hydrogen bond acceptor (HBA) strength, thus demonstrating that retention is not solely based on hydrophobicity. A similar result was observed for a C14TAB-MEKC system, however, the HBA characteristic of C14TAB selectively differentiates between the solutes with different HBD strength. In addition, quantitative retention-activity relationships in MEKC were also investigated for 9 corticosteroids. Two types of biological activities [small intestinal absorption in the rat (log A/NA) and protein binding to human serum albumin (log B/F) were examined in this work. High correlations were observed between bioactivity and log k' in MEKC using bile salt surfactants and mixed bile salt systems.

Adrenal Cortex Hormones↗

PubMed1996

Structure-retention relationships of steroid hormones in reversed-phase liquid chromatography and micellar electrokinetic capillary chromatography.

Quantitative structure-retention relationship (QSRR) studies are useful in retention prediction, finding the relevant structural descriptors for analytes and estimating the relative biological activities of a series of analytes. Most of the studies have been conducted by RP-HPLC and a few by micellar electrokinetic capillary chromatography (MECC). The aim of this work was to find structural parameters and characteristics related to the RP retention and electrophoretic migration of steroids in order to predict the retention/migration of steroid hormones on the basis of their molecular structure. Retention data were obtained with an ODS column using a mobile phases aqueous methanol-acetonitrile (mobile phase A) and methanol-tetrahydrofuran (mobile phase B). MECC was conducted with a sodium dodecyl sulfate (SDS)-borate system and with a mixed micellar solution of SDS and sodium cholate. Several topological indices, such as the connectivity indices, chi, were used as structural parameters. Steric factors seem to have a great effect on the retention of steroid hormones, especially with the MeCN-containing mobile phase. Retention in mobile phase B could be predicted more accurately. Topological indices can be used in the modelling and prediction of the retention/migration of steroid hormones when the solutes form a congeneric series and stereochemical properties do not govern the separation process.

Chromatography↗

PubMed1996

Chemical selectivity in micellar electrokinetic chromatography. II. Rationalization of elution patterns in different surfactant systems.

Retention behavior in micellar electrokinetic chromatography (MEKC) is investigated using linear solvation energy relationships (LSERs) for two pseudo-stationary phases, one consisting of cationic micelles of tetradecyltrimethylammonium bromide (C14TAB) and the other of an anionic triblock copolymer, poly(methyl methacrylate-ethyl acrylate-methacrylic acid) (Elvacite 2669). It was found that solutes' migration behaviors in these two MEKC systems are mainly influenced by their size (V/100) and hydrogen bonding acceptor (HBA) strength (beta). However, solutes' hydrogen bonding donor (HBD) strength (alpha) has minor effects on their migration in MEKC. The characteristics of these two systems were compared to three other previously reported anionic micellar systems of sodium dodecyl sulfate (SDS) (anionic hydrocarbon), sodium cholate (SC) (anionic bile salt) and lithium perfluorooctane sulfonate (LiPFOS) (anionic fluorocarbon). It was concluded that hydrogen bonding interactions play a major role in providing different chemical selectivity among these five MEKC systems. Both C14TAB micelles and the ionic polymer of Elvacite 2669 provide hydrogen bonding acceptor (HBA) sites for solutes, which is similar to SC micelles. In fact, C14TAB is the strongest HBA, while Elvacite 2669 has HBA strength similar to that of SC micelles. On the other hand, the fluorocarbon micelles of LiPFOS are the strongest hydrogen bond donor (HBD) micelles, followed by the weak HBD SDS micelles. In general, cavity formation has little or no effect on chemical selectivity among hydrocarbon surfactant MEKC systems (i.e., SDS, SC and C14TAB). Information obtained from the LSER analysis is used to rationalize the elution patterns in MEKC with different types of pseudo-stationary phases.

Chromatography, Liquid↗

PubMed1981

An improved method for the isolation of the major protein of the gonococcal outer membrane in an antigenically reactive form.

The major outer membrane protein (protein I) has been isolated from Neisseria gonorrhoeae strain P9 in an immunologically reactive form. Membranes were sequentially extracted with the detergents sodium cholate and Empigen BB. Protein I was enriched in the Empigen-soluble fraction and was separated from other proteins and lipopolysaccharide by gel filtration chromatography on Sephadex G-200. The purified protein retained its antigenic activity with antiserum raised against the unfractionated outer membrane complex.

Antigens, Bacterial↗

PubMed1986

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

3-Hydroxysteroid Dehydrogenases↗

PubMed1987

Further evidence that there is more than one adrenal 21-hydroxylase system.

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.

17-alpha-Hydroxypregnenolone↗

PubMed1984

Toxic effects of intrahepatic reflux of monooctanoin in a canine model.

Monooctanoin (MO), when infused into the common bile duct (CBD), is an effective agent in dissolving retained CBD stones. If the stone migrates and obstructs the distal CBD, the solution could be infused under pressure resulting in intrahepatic reflux. The relationship between the infusion pressure and the safety of monooctanoin has not been evaluated. To study the effects of intrahepatic reflux of MO, a canine model was used and solutions were infused into an obstructed CBD under controlled pressure. Solutions of normal saline (NS), 150 mM sodium cholate (Ch), or MO were infused under pressures of 30, 40, and 50 cm. All of eight dogs died when infused with MO at 50 cm pressure with a mean administered dose of 1.4 +/- 0.4 cc/kg within a mean time of 87 +/- 38 min. Three of four dogs died at 40 cm pressure (1.7 +/- 0.4 cc/kg; 133 +/- 95 min) and only one of three dogs died at 30 cm pressure (2.5 cc/kg; 335 min). These dogs died from progressive hypoxia, acidosis, hemolysis, and hemorrhagic pneumonitis. Four dogs each were administered Ch at 50 and 40 cm pressure and all died with an average absorption of 19 cc/kg. Six dogs were tested with NS at 50 cm and all survived despite absorbing 180 cc/kg in 6 hr. MO at 50 cm pressure and Ch at both 40 and 50 cm pressure were significantly more toxic than saline. It is concluded that MO and Ch infused under pressure into CBD carry a significant risk of serious side effects. The infusion pressure must be monitored to prevent increased biliary pressure which might lead to intrahepatic reflux.

Animals↗

PubMed1983

Binding of phencyclidine to the detergent solubilized acetylcholine receptor from Torpedo marmorata.

The binding of phencyclidine to the acetylcholine receptor from Torpedo marmorata electroplaque was measured following solubilization of the receptor in sodium cholate followed by the exchange of cholate for Tween 80. In both the membrane-bound and solubilized AChR, the addition of cholinergic agonists simultaneously with the addition of PCP results in a 100 to 1000 fold increase in the PCP association rate and a 5 to 10 fold increase in the dissociation rate as compared to the unliganded AChR or AChR equilibrated with agonist prior to PCP addition. In addition, the number of binding sites and the pharmacological properties of the binding are not markedly changed in the soluble receptor. These results suggest that the acetylcholine receptor can undergo similar conformational transitions in the membrane-bound and the Tween 80 solubilized form and that phencyclidine can monitor these transitions in both cases.

Animals↗

PubMed1983

Effect of Clostridium butyricum on the formation and dissolution of gallstones in experimental cholesterol cholelithiasis.

The effect of oral administration of Clostridium butyricum Miyairi on the formation and dissolution of gallstones was examined in a mouse model of cholesterol cholelithiasis, in comparison with that of chenodeoxycholic acid (CDCA). A diet containing cholesterol and sodium cholate was used as a lithogenic diet. The feeding of the lithogenic diet containing 1.0 X 10(8) cells of this bacterium per g for five weeks prevented the formation of gallstones, resulted in lower values of the gallstone index, incidence and cholesterol content of gallstones than those of untreated group by 46, 43 and 46%, in order. Furthermore, after mice were maintained on the lithogenic diet for five weeks, subsequent bacterial treatment exhibited a marked gallstone dissolution effect. The feeding of 10(8) cells/g was effective for the prevention of gallstone formation and dissolution of gallstones, but that of 10(6) cells/g was not significantly effective. The gallstone index of the bacterium-treated group was lower than that of CDCA-treated group during the period of experiment, but there was no difference in the cholesterol content of gallstones between these two groups, which suggests a different mechanism of action of the bacterium from that of CDCA.

Animals↗

PubMed1992

Mutagenic activation of aflatoxin B1 by pulmonary, renal, and hepatic cytochrome P450s from rats.

The genotoxic and mutagenic activation of aflatoxin B1 (AFB1) by hepatic, renal, and pulmonary microsomes and purified cytochrome P450s was investigated in Salmonella typhimurium TA1535/pSK1002 cells in which an umu response shows DNA damage. The activity of the hepatic microsomes was greatest. Pulmonary microsomes had moderate activity and renal microsomes had low activity. P450 2C11, 2B1, 3A2, 4A2, 4B1, K-2, and K-4 were assayed in a reconstituted system with dilauroylphosphatidylcholine (DLPC). P450 2C11 (a major hepatic cytochrome P450 in male rats) had high activity. P450 2B1 (a major form as well as P450 4B1 in pulmonary microsomes) and K-2 (a minor form in renal microsomes) had moderate activity. P450 4A2 (a major form in renal microsomes), P450 K-4 (a renal form), and P450 4B1 had low activity. P450 3A2 did not have high activity in these conditions but it had high activity toward AFB1 in a modified reconstituted system with a lipid mixture and sodium cholate instead of DLPC only. The activities of other forms were not enhanced by the modification of reconstituted system. Anti-P450 2C11 or 3A2 antibodies inhibited the bioactivation of AFB1 by hepatic microsomes to 50%. These results suggest that the greater ability of hepatic microsomes as compared with pulmonary and renal microsomes to metabolize AFB1 to mutagenic products is a function of the relative proportions of the highly active cytochrome P450s, P450 2C11 and 3A2, in the liver.

Aflatoxin B1↗

PubMed1996

The effects of pH and bile salts on the binding of MeIQx to wheat bran fibre.

The effects of pH and of the bile salts, sodium cholate, chenodeoxycholate, taurocholate, deoxycholate and glycodeoxycholate on the adsorption of the heterocyclic aromatic amine mutagen and carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) onto wheat bran cell wall material have been examined. The highest binding affinity of MeIQx for bran was at pH 5.5. Binding affinity declined more rapidly with pH > 5.5 than with pH < 5.5. Bile salts in solution did not appreciably affect the binding of MeIQx to bran, but where the bile salts formed a suspension then the adsorption was reduced. Where precipitation of bile salts occurred then the free concentration of MeIQx was also reduced, indicating that MeIQx binding/immobilization could be enhanced through interaction with bile salts.

Bile Acids and Salts↗

PubMed1983

A rapid sensitive radioimmunoassay for rhodopsin: development and application.

Antisera have been raised against unbleached chromatographically pure bovine rhodopsin. A rapid sensitive radioimmunoassay (RIA) has been developed for rhodopsin, employing [125I]rhodopsin labeled using the Bolton-Hunter reagent. Protein A-bearing Staphylococcus aureus cells are used to precipitate the immune complex. Subpicomolar amounts of rhodopsin can be determined when the RIA is performed in the dark using any of nine different detergents tested. When the RIA is performed using bleached rhodopsin, the results are dependent upon the detergent in which the assay is performed. Bleached rhodopsin is most immunologically similar to unbleached rhodopsin in the mildest detergents and less similar in harsher detergents. One and a half times more bleached rhodopsin is required to compete to the same extent as unbleached rhodopsin when the RIA is performed in digitonin. Results for other detergents are: sucrose monoester, 5.0; CHAPS, 64; sodium cholate, 91; octylglucoside, 250; Triton X-100, 430; Emulphogene, 570; Ammonyx LO approximately 14,000; CTAB, unmeasureable. Other species of rhodopsin were tested as competitive in the RIA. Pig rhodopsin is 1/100th as effective a competitor as bovine rhodopsin, rat 1/200th, and frog 1/800th.

Animals↗

PubMed1990

Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes.

Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.

Animals↗

PubMedDate not supplied

Irreversible labelling of delta-opioid receptors in rat brain and neuroblastoma cells by [3H]azido-DTLET: characterization of subunits and autoradiographic visualization of the covalent binding.

[3H]Az-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr), a photoaffinity probe for delta opioid receptors binds to a single class of sites in rat brain membranes with a high affinity (KD = 1.66 nM). The selectivity index of Az-DTLET (KI delta/KI mu = 0.036) is better than that of its precursor DTLET (0.053). Rat brain or neuroblastoma glioma cells membranes were incubated with 10 nM [3H]Az-DTLET, washed and irradiated with U.V. After irradiation a fraction (20-30%) of specific binding was found to remain indissociable after 10 min at 60 degrees C and was considered as irreversible. This fraction increased as a function of the irradiation time. The radioactivity irreversibly bound to rat brain membranes, solubilized by sodium cholate, was associated with high molecular weight species (200,000 daltons). In denaturing conditions (SDS 2%), the [3H]Az-DTLET specific binding was associated with molecular components of 45-50 K and 90-100 K daltons. In contrast, when opioid receptors were prelabelled by [3H]Az-DTLET, solubilized by Na-cholate and irradiated, the radioactivity was only recovered with subunits of 45-50 K daltons. The autoradiographic localization of the irreversibly bound [3H]Az-DTLET in rat brain was identical to that of reversibly bound [3H]DTLET or [3H]Az-DTLET. These results suggest that [3H]Az-DTLET represents an adequate specific probe for studies on the structure, function and anatomical distribution at light and even electron microscopic level of delta-opioid receptors.

Animals↗

PubMed1994

Apolipoprotein E self-association in solution studied by non-radiative energy transfer.

The self-association of human apolipoprotein E (apoE), isolated from plasma very low density lipoproteins, was studied at apoE concentrations less than 0.6 microM by non-radiative energy transfer. ApoE was separately labeled with a fluorescent donor group i.e. dansyl chloride (apoE/D) and with an acceptor i.e. fluorescein isothiocyanate (apoE/F). Mixed apoE/D:apoE/F complexes were prepared either by incubation or the donor- and of the acceptor-labeled apoE or by renaturation during dialysis of the apoE/D:apoE/F mixture pre-denatured by addition of guanidine hydrochloride or by treatment with sodium cholate. The efficiency of energy transfer E at an equimolar ratio of the donor to acceptor and a ratio of 1.9 mol fluorescein/mol protein amounted to 29.2 +/- 2.6% (n = 3). The E value increased linearly with increasing acceptor fraction in the mixture. The state of self-association of apoE as tetramers within this concentration range was confirmed by cross-linking experiments with a water-soluble bifunctional reagent. This approach can be applied to the study of protein-protein interactions in apolipoprotein-phospholipid recombinants.

Apolipoproteins E↗