Aqueous Buffer System Research Articles

Binding, Uptake, and Transport of Hypericin by Caco-2 Cell Monolayers

□ The biological evaluation of hypericin in various test models is hampered by its very poor water solubility. In the present study cyclodextrin formulations and liposomal preparations were investigated for improved delivery and solubility of hypericin in aqueous buffer systems. Caco-2 cells, grown to tight monolayers on 96-well tissue culture plates as well as on Transwell polycarbonate filters, were used to study the membrane binding and the epithelial transport of hypericin. Cumulative transport of hypericin, which could not be measured without the use of cyclodextrins, in apical-to-basolateral direction from cyclodextrin–hypericin buffer solutions was 3–5% at 37 °C and approximately 0.12% at 4 °C after 5 h. After an incubation time of 1 h at 37 and 4 °C, 12.7%±2. 6% and 6.5%±0.8%, respectively, of hypericin were found to be bound to or taken up by Caco-2 cells. Liposomal formulations markedly increased the solubility of hypericin in Krebs–Ringer buffer, but there was no effect observed on the binding and transport of hypericin delivered by liposomes in the Caco-2 cell model. Due to the fluorescence properties of hypericin, its interaction with the cells could be visualized by confocal laser scanning microscopy. The results indicate that a significant accumulation of the drug in the cell membrane and the cell nucleus membrane takes place. We conclude that hypericin is absorbed through the intestinal epithelium by passive transcellular diffusion and that increasing its solubility by cyclodextrin appears as a promising approach to increase its oral bioavailability for pharmaceutical formulations.

Thermodynamics of Micelle−Water Partitioning in Micellar Electrokinetic Chromatography: Comparisons with 1-Octanol−Water Partitioning and Biopartitioning

Micellar electrokinetic chromatography (MEKC) was evalu ated as a model for biopartitioning. The thermodynamics for water−micelle partitioning are measured and compared with literature values for both biopartitioning and water−1-octanol partitioning. It was found that the free energy of transfer (ΔG) is dominated by the entropic term (ΔS) for both water−micelle partitioning and biopartitioning, but the enthalpic term (ΔH) dominates for water−1-octanol partitioning. Thermodynamic values of transfer and water−micelle partition coefficients are presented for a series of 67 solutes with varying functionalities. This work demonstrates the usefulness of MEKC for estimating biopartitioning by establishing a correlation between biopartitioning and water−micelle partitioning. Also, this method incorporates the economic advantages of capillary electrophoresis (CE), which include fast analysis times, low sample consumption, the use of aqueous buffer systems, and low organic solvent use and disposal. Solute retention...

Capillary zone electrophoresis in organic solvents: separation of anions in methanolic buffer solutions

A tris(hydroxymethyl)aminomethane-acetate buffer system with methanol as solvent has been used at an apparent pH of 8.5 for the separation of six aromatic and aliphatic acids. Compared to pure aqueous buffer systems with various pH values (4.4, 5.5, 6.2 and 8.0) improved separation was obtained due to increased selectivity. This improvement is related to a specific shift in the p K a values of the solutes in the organic solvent, and is interpreted by the concept of the transfer activity coefficient and the medium effect.

Protection of plasmid pBR322 DNA by flavonoids against single-strand breaks induced by singlet molecular oxygen

Flavonoids, the dominant colouring pigments of plants, as well as the related polyphenol tannic acid significantly inhibit single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen ( 1O 2). This reactive species of oxygen was generated in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate. Among the antioxidants examined, myricetin showed the highest protective ability, followed by tannic acid, (+) catechin, rutin, fisetin, luteolin and apigenin, when the inhibitory abilities were compared at 90 min after incubation. The protective abilities of these compounds were both time and concentration dependent. At equimolar concentrations (100 μM) the antioxidant effect of myricetin was better than that of other known antioxidants such as lipoate, α-tocopherol and β-carotene. Data, when analysed in relation to the structures of various compounds, showed a rough correlation with protective abilities. Owing to the abundance of these compounds in our normal diet, they may play significant roles in preventing oxidative damage resulting from potentially deleterious 1O 2.

Nitro radical anion formation from nifurtimox II: electrochemical evidence

The cyclic voltammetric (CV) behaviour of nifurtimox was studied. The addition of aprotic solvent (DMF) to nifurtimox in an aqueous citrate buffer system was examined. Qualitatively this results in separation of the initial irreversible four-electron reduction into two stages, the RNO 2/RNO 2 .− and RNO 2 .−,4H +/RNHOH,H 2O couples respectively. Particul attention was directed to the one-electron RNO 2/RNO 2 .− couple as measured by the CV mode in mixed media. Analysis of the CV response as a function scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second-order kinetic with a stability constant of 954 L mol −1 s −1 and a half-life of 1.05 s for 1 mM nifurtimox in aqueous citrate buffer, pH 10.5, + DMF, 40:60.

Diminution of singlet oxygen-induced DNA damage by curcmin and related antioxidants

Curcumin, the natural antioxidant from turmeric, an Indian spice, and its derivatives have significant abilities to protect plasmid pBR322 against single-strand breaks induced by singlet oxygen ( 1O 2), a reactive oxygen species with potential genotoxic/mutagenic properties. 1O 2 was generated at 37°C in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate (NDPO 2). Among the compounds, tested, curcumin was the most effective inhibitor of DNA damage followed by desmethoxycurcumin, bisdesmethoxycurcumin and other derivatives. The observed antioxidant activity was both time-and concentration-dependent. The protectice ability of curcumin was higher than that of the well-known biological antioxidants lipoate, α-tocopherol and β-carotene. However, the highest protective ability with saturating concentrations of curcumin did not exceed 50%. The ability of curcumin and its derivatives to protect DNA against 1O 2 seems to be related to their structures and may at least partly explain the therapeutic and other beneficial effects of these compounds including anticarcinogenic and antimutagenic properties.

Kinetic study on the nitrosation of dibenzylamine in a model system

A kinetic study of the formation of N-nitrosodibenzylamine (NDBzA), from the nitrosation of dibenzylamine (DBzA) by sodium nitrite, was performed in a model system under conditions (temperature, pH) that are similar to those encountered in the industrial production of hams processed in elastic rubber nettings. The nitrosation reaction was carried out in a KH 2PO 4 buffer (0.5 m) at pH 5.8 and at a temperature of 69 C. Since DBzA is insoluble in an aqueous buffer system, a non-ionic surfactant. Tween 20, was used as a solubilizing agent. The nitrosation reaction exhibited first-order kinetics with respect to DBzA and second-order kinetics with respect to nitrite. The calculated rate constant was 4.7 ± 0.5 m −2/ min. The pH profile of NDBzA formation was also determined. The optimal pH of NDBzA formation, 3.12, was close to the pK a of nitrous acid (HNO 2, pK a = 3.1).

A nitro radical anion formation from nifedipine: an electrochemical approach

The cyclic voltammetric behaviour of nifedipine was studied. The addition of three aprotic solvents to nifedipine in an aqueous citrate buffer system was examined. Qualitatively they result in separation of the initial irreversible 4 electron reduction into two stages, the NO 2/RNO . 2 − and RNO . 2 −, 4H +/RNHOH, H 2O couples, respectively. Particular attention was directed to the 1-electron RNO 2/RNO . 2 − couple as measured by the cyclic voltammetric mode in mixed media. Analysis of the cyclic voltammetric response as a function of scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second order kinetics with a stability constant of 1.1 × 10 −3 l mol −1 s −1 and a half-life time of 0.09 s for 1 mM of nifedipine in aqueous citrate buffer, pH 7.4/DMF; 50:50.

New fluorescent labels for time-resolved detection of biomolecules

New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of "multiplex dyes," we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.

Chloroperoxidase-mediated modifications of petroporphyrins and asphaltenes

Octaethylporphine, nickel octaethylporphine, vanadyl octaethylporphine, and a petroporphyrin-rich, low-molecular-weight fraction of asphaltenes from Cold Lake heavy oil were treated with chloroperoxidase from Caldariomyces fumago. Reactions in aqueous phosphate buffer (pH 3.0) or in a ternary solvent system of toluene, isopropanol, and water (3 m m phosphate buffer, pH 3.0) were absolutely dependent on the presence of hydrogen peroxide and chloride. Enzyme treatment resulted in reduction of the absorption of the Soret peak. The porphyrins and asphaltenes were insoluble in the aqueous buffer system; thus, mass transfer limited the reactions. These substrates were more soluble in the ternary system and the reactions were more complete, yielding decreases in metal recovery associated with the methylene chloride-soluble porphyrin-containing material. These decreases were: 93% of the Ni from nickel octaethylporphine, 53% of the V from vanadyl octaethylporphine, and 20% of the total Ni and V from the asphaltene fraction. This work clearly demonstrated that an extracellular enzyme, chloroperoxidase, can alter components in the asphaltene fraction of petroleum. Because of the requirement for chloride, the enzyme-mediated reactions likely yield chlorinated products which would be undesirable in a refinery feedstock if this enzymatic process was used for the demetallation of petroleum.

Immobilized phenylboronic acids for the selective extraction of β-blocking drugs from aqueous solution and plasma

The use of phenylboronic acid (PBA) solid-phase extraction cartridges for the extraction of a range of β-blockers from aqueous buffer and plasma has been investigated and compared with other phases commonly used for solid-phase extraction. PBA was found to provide an efficient means for the extraction of this class of compound from simple aqueous buffer systems. Extraction from buffers was pH-dependent and gave optimum results at approximately pH 8. Extraction from plasma was less efficient for some of the test compounds; this matrix effect was probably due to protein binding. Chromatography showed that plasma extracts were free of major sources of interference.

Evaluation of N-hydroxy-2-thiopyridone as a nonmetal dependent source of the hydroxyl radical (HO·) in aqueous systems

N-hydroxy-2-thiopyridone (1), an established source of the hydroxyl radical ( HO·, Boivin, J., Crepon, E., and Zard, S. Z. (1990), Tetrahedron Lett. 31, 6869–6872), produced HO· under conditions directly applicable to biological studies. Generation of HO· by subjecting 1 to irradiation with visible light was monitored in the following “HO·” assays: deoxyribose degradation, addition to dimethyl sulfoxide, and hydroxylation of salicylate and phenol. All four assays demonstrated the production of HO· from 1 (added as a sodium salt) under mild conditions in aqueous buffer systems. An improved analysis method was developed for the phenol assay. A time course analysis demonstrated that a flux of HO· is generated from 1 throughout the irradiation period, in contrast to the classical Fenton reaction of H 2O 2 with a transition metal in which a burst of HO· is generated in a short time period. While a thiyl radical is generated from 1 concurrent with HO· generation, this species does not contribute to, or interfere with, any of the HO· assays, suggesting that it is weakly reactive in aqueous buffers. Thus, irradiation of 1 can be used as an alternative, complementary, approach for the un-equivocal generation of the biologically significant and reactive HO·.

Microemulsions as reaction media for immobilization of proteins to hydrophilized surfaces

The effect of microemulsion as reaction medium for immobilization of proteins to polystyrene hydrophilized with poly(ethylene glycol) or polysaccharide has been investigated. The amount of albumin, immunoglobulin G (IgG) and collagen immobilized from microemulsion was increased 10–100 times, compared with immobilization from an aqueous buffer system. The stability of anti-IgG and interleukin-2 was studied in different microemulsion systems. Anti-IgG was found to be stable in microemulsions based on sodium bis(2-ethylhexyl)sulphosuccinate (AOT) and C 12EO 5, whereas the non-ionic surfactant di-C 9φEO 9 almost instantly destroyed the antigen-binding properties. Interleukin-2 completely lost its biological activity in an AOT-based microemulsion. We have also found that the choice of microemulsion influences coupling efficiency of the protein. An AOT-based system is preferred for immobilization of mycoplasma antibody, while a microemulsion based on C 12EO 5 is preferred for borrelia antigen.

Quantitative Analyses of Hydrophobicity of Di- to Pentapeptides Having Un-ionizable Side Chains with Substituent and Structural Parameters

With experimental conditions established recently, we measured the partition ratio (P) of 124 di- to pentapeptides composed of amino acids having un-ionizable side chains in a 1-octanol:pH 7.0 aqueous phosphate buffer system as an approximate zwitterionized “molecular” partition coefficient (P). Empirical equations of good quality, correlating the variations in log P value of peptides with free energy-related physicochemical parameters for the side chain substituents and substructures, were formulated. The significant side chain parameters were those representing the intrinsic hydrophobicity, the steric effect on the relative solvation of functional groups on the backbone, and the conformational potential index derived from the Chou-Fasman β-turn potential parameters. For polar side chains, specific indicator variables were required for intramolecular hydrogen-bond formations and the “polar proximity effect” for reductions of hydrophilicity observed when polar groups are crowded together. The proline residue was shown to contribute to the log P value depending not only on its location on the backbone but also on the number of residues in peptides. On the basis of the analyses, we proposed a new “effective” hydrophobicity index for un-ionizable side chains which could predict the secondary structure of oligopeptides.

Phase Separation Coupled with Gelation in Polyethylene Glycol–Gelatin–Aqueous Buffer System

Abstract Phase behavior of a pseudoternary system, gelatin–polyethylene glycol–acetate buffer, was examined. Phase diagram was constructed by determining the compositions of separated phases. One phase contained mostly gelatin but a trace amount of polyethylene glycol. When temperature was lower than the gelation temperature corresponding to the composition of the separated phase, diverse outlooks of the whole system were found. Observation of solutions under an optical microscope revealed microgels or network structure when gelatin was labeled with sirius red. Two different types of microgel formation were noted: nucleation-growth and spinodal decomposition. Size of the microgels formed by spinodal decomposition decreased with quench temperature.

Axial-beam absorbance detection for capillary electrophoresis

A novel approach to absorbance detection has been demonstrated for capillary eletrophoresis utilizing an incident light beam directed along the capillary axis. The resulting absorption path length is roughly equal to the width of the sample bands present. This represents a 60-fold improvement in analytical path length over conventional on-column absorbance detectors withourt any loss of separation efficiency. The method can be used with completely aqueous buffer systems or those containing organic modifiers, and is applicable to both fused-silica and PTFE capillaries.

Electrochemical Characteristics of Nitroheterocyclic Compounds of Biological Interest VI. The Misonidazole Radical Anion

The addition of four aprotic solvents to misonidazole in an aqueous buffer system has been examined electrochemically. Qualitatively they all result in separation of the initial irreversible 4 electron reduction step into two stages, the RNO2/RNO2- and RNO2-/RNHOH couples respectively. Despite some difficulties in achieving measurements for the discrete RNO2/RNO2- without interference from the following reduction step, it was clear that the various aprotic solvents influenced the lifetime of the RNO2- species to different degrees. Resolution of the two processes was best achieved using a water-acetone system and this has been employed to study the lifetimes of the misonidazole radical anion as a function of acetone content and drug concentration. Analysis of the cyclic voltammetric response showed a second order decay pathway, in line with the metronidazole system studied under similar conditions. This has been compared with results from pulse radiolysis work, which suggested a first order reaction of unknown pathway for 2-nitroimidazole radical anions.

Hydrophobicity of N‐Acetyl‐Di‐ and Tripeptide Amides Having Unionizable Side Chains and Correlation with Substituent and Structural Parameters

AbstractThe log P value of 53 N‐acetyl‐di‐ and tripeptide amides composed of amino acids having unionizable side chains was measured in a 1‐octanol/pH 7.0 aqueous buffer system. The factors governing the variations in the log P value among these protected peptides were quantitatively analyzed to formulate a correlation equation with free‐energy‐related physicochemical and substructural parameters. The log P value was governed by the sum of the hydrophobicity of side chains and the backbone as well as by the steric effects of side chain substituents on the relative solvation of the backbone CONH groups. The log P value was found to decrease by 0.6 log unit for the peptide bond, other factors being equal. For amino acids with polar side chains, the log P value was also affected by the “polar proximity factor” and/or intramolecular hydrogen bond formation in a way similar to that of zwitterionized peptides reported previously.

Electrochemical characteristics of nitro-heterocyclic compounds of biological interest. II. Nitrosochloramphenicol.

The electrochemical characteristics of nitrosochloramphenicol have been studied in aqueous buffer systems (pH 7.1) using direct current (d.c.) and differential pulse polarography, cyclic voltammetry and coulometric techniques. Up to 4 charge-transfer steps can be identified. The first reduction step is reversible both chemically and electrochemically, the charge-transfer product showing no tendency to undergo further reaction on the electrochemical time-scale. In contrast, the second reduction step is irreversible, with the product undergoing a fast following reaction to yield a redox-active species which was detected by cyclic voltammetry. From the data and by comparison with related systems, two reduction mechanisms are possible and are discussed.

Hydrophobicity of Di‐ and Tripeptides Having Unionizable Side Chains and Correlation with Substituent and Structural Parameters

AbstractAfter establishing experimental conditions to obtain a reliable partition ratio, P', we measured the value of 59 di‐ and tripeptides composed of amino acids having unionizable side chains in a 1‐octanol/pH 7.0 aqueous phosphate buffer system in order to approximate the true partition coefficient. We then formulated an equation correlating the variations of log P' (pH 7) among peptides with free‐energy‐related physicochemical parameters for the side chain substituents and substructures. The log P' value of di‐ and tripeptides is governed by the sum of the hydrophobicity of the peptide backbone and side chains as well as by the steric effect of side chain substituents on the relative solvation of backbone and terminal functional groups. For peptides containing amino acids with polar side chains, the log P' value is higher than that predicted by these effects. The difference is larger as the polar heteroatom in side chains gets closer to the backbone. This may correspond to the “polar proximity factor” for reductions of hydrophilicity observed when polar groups are crowded together. For the side chain of serine and threonine, the difference is even higher than that expected by the polar proximity effect, presumably due to intramolecular hydrogen bond formation.