Effect of structure modification of chondroitin sulfate C on its enantioselectivity to basic drugs in capillary electrophoresis

The effect of structure modification of chondroitin sulfate C on its enantioselectivity to several representative basic drugs in capillary electrophoresis was investigated. Chemical desulfation showed no remarkable decrease in selectivity, whereas depolymerization with chondroitinase ABC resulted in complete loss of selectivity. Comparison with chondroitin sulfate A indicated considerable decrease in selectivity with this isomer. The great retention of enantioselectivity in the desulfated derivative suggests that the selectivity comes from the difference of the magnitude of an interaction in the multipoint mechanism between a part of the drug molecule and a functional group in chondroitin sulfate C other than the sulfate group. The sulfate group is not considered to play a major role for chiral separation. The complete loss of selectivity by depolymerization is consistent with a general tendency of lower selectivity in smaller saccharides, and the priority of chondroitin sulfate C to chondroitin sulfate A suggests the importance of the hydroxyl group at C4 in the galactosamine residue. During the course of this work we observed heavy tailing of the peaks of basic drugs in some batches of uncoated fused-silica capillaries under acidic conditions and solved this problem by doubly coating capillaries with Polybrene followed by chondroitin sulfate C. On the other hand, we demonstrated the usefulness of a special technique which uses a short, wider bore PTFE tube-attached capillary for the study of the effect of depolymerization, in order to minimize sample amount.     

The influence of chondroitin sulfate on composite multilamellar liposomes containing chitosan

A composite multilamellar liposome containing chitosan attached to the inside and outside of the membrane as well as an opposite charged polyelectrolyte, chondroitin, adsorbed at the surface was developed. Not only the chitosan/chondroitin ratio but also the concentration of them were varied. The structure and superficial properties of the liposomes were studied through a combination of light scattering, zeta potential, and small-angle X-rays scattering techniques. While the chitosan/chondroitin ratio affected the superficial charge distributions, the concentration of polyelectrolytes affected the structural properties of the liposomes, as the rigidity of the phospholipid layers. The superficial charge of the resultant composite liposome was influenced by the type and concentration of the polyelectrolyte. Information about the charge density could be obtained by the treatment of zeta potential data, and it was used to estimate the amount of chondroitin adsorbed to the liposome surface. Applying the modified Caillé theory to the X-rays scattering curves, information about the internal structure of the liposomes was accessed. The ability to control the properties of composite multilamellar liposomes is an important issue when they have to be applied as a biomaterial device component.     

Action of surface-active substances on biological membranes I. Effect of chemical modification of membranes on hemolysis of erythrocytes by sodium alkyl sulfates

Summary The hemolytic action of a homologous series of sodium alkyl sulfates (C ₈ toC ₁₅) on dog and human erythrocytes was measured. The influence of trypsin, pronase and neuraminidase treatments on the lytic resistance of human red cells was studied. A new approach evaluating the hemolytic activity of ionic detergents is expressed as a ratio of a concentration providing 50% lysis to its CMC-value. The results obtained are examined for their bearing on the use of surface-active agents as a means to study the structure organization of biological membranes.

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Zusammenfassung Es wurde die hämolytische Wirkung homologer Reihen von Natriumalkylsulfaten an Erythrozyten von Mensch und Hund gemessen. Um die hämolytische Aktivität der ionogenen Detergenzien zu charakterisieren, wurde das Verhältnis der Konzentration, welche eine 50%-ige Lyse bewirkt, zum cmc-Wert gewählt.

     

Study of the effect of dose-rate on radiation-induced damage to human erythrocytes

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%) were irradiated with γ-rays at three dose-rates of 66.7, 36.7, 25 Gy min⁻¹ in order to investigate the influence of the dose-rate on radiation-induced membrane damage, hemoglobin oxidation and loss of reduced glutathione.The obtained results showed that such processes as erythrocyte hemolysis, lipid and protein destruction depend on the radiation dose-rate. The parameter values describing these processes showed an inverse dose-rate effect.     

Effects of a high alpha-linolenate and high linoleate diet on hemolysis and lipid peroxidation of rat erythrocytes.

alpha-Linolenic acid (18:3n-3) is known to autoxidize several fold faster than linoleic acid (18:2n-6). Feeding a high alpha-linolenate or a high linoleate diet to rats resulted in significant changes in the n-3/n-6 ratios of 20 and 22 carbon highly unsaturated fatty acids in erythrocytes. However, the rates of hemolysis observed in N2- or O2-atmosphere were similar between the two dietary groups. No significant amounts of conjugated dienes were detected and no measurable changes in the fatty acid compositions were observed during the incubations, indicating that the hemolysis occurred without involving significant lipid peroxidation. When stimulated with a free radical initiator, [2,2'-azobis-(2-amidinopropane)dihydrochloride] (AAPH), hemolysis occurred more rapidly, conjugated dienes formed and unsaturated/saturated ratios of phospholipid fatty acids decreased. However, no statistically significant difference was observed in these parameters of the two dietary groups. These results indicate that hemolysis occurs without involving lipid peroxidation but is accelerated by free radicals through lipid peroxidation, and that the difference in autoxidizabilities of alpha-linolenate and linoleate is not reflected in the rates of hemolysis and autoxidation in rat erythrocytes.     

Effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on human monocytic THP-1 cell migration

Angiogenesis serves as a crucial factor in disease development and progression, such as cancer metastasis, and monocyte migration is one of the key steps for angiogenesis. Therapeutic modulation of angiogenesis is a promising new therapeutic avenue under investigation. In this study, effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on monocyte migration were investigated. Human monocytic THP-1 cells were from Riken Cell Bank (Tsukuba, Japan) and vascular endothelial cells (VECs) were obtained from swine thoracic aorta. The migration experimental system was adapted from Falcon™ Cell Culture Inserts with pore sizes of 3 and 8 μm cultured endothelial cells or not on the insert polyethylene terephthalate (PET) membranes. Four VEGF concentrations (0, 10, 50 and 100 ng/ml) and three concentrations of chondroitin sulfate A (0, 1.25 and 5.0 mg/ml) were used to investigate their effects on THP-1 cell migration ability through PET membranes and VECs monolayer. The THP-1 cell migration was evaluated by counting the number of migrated cells related to the total number of cells under a microscope. We counted the migration cells every 1 h on a Tatai-type hemocytometer using an inverted microscope for total 7 h. For inserts with pore sizes of 3 and 8 μm, the THP-1 cell migration increased with VEGF concentrations; however, cell migration decreased with the chondroitin sulfate A concentration. Our results demonstrated that VEGF accelerated monocyte migration through endothelial monolayer and chondroitin sulfate A is an effective inhibitor of monocyte migration for angiogenesis.     

Preparation of finely dispersed drugs. III. Cyclosporine

Two different precipitation processes are described, which produced dispersions of spherical particles of cyclosporine ranging in diameter from approximately 10 nm to several micrometers. This drug is of interest for its immunosuppressive activity in the antirejection of transplanted organs. The effects of several experimental parameters on the average particles size and uniformity have been investigated. Aging of spherical particles resulted in large crystalline-type aggregates.     

Effect of phenylhydrazine-induced structural alterations of human erythrocytes on basic drug penetration

The phospholipid organization, lipid fluidity and spectrin degradation were measured in human erythrocytes oxidized with phenylhydrazine, and the contribution of these structural alterations to the penetration of perazine and promethazine into the membrane was estimated. It was found that exposure of erythrocytes to phenylhydrazine (0.2--0.4 mg/ml) produced a 35--40% decrease in the amount of spectrin, with resultant gross morphological changes to the echinocyte conformation. The phosphatidylethanolamine content in the treated erythrocytes was greatly lowered compared with that in the untreated cells. Treatment with phenylhydrazine (0.05--0.2 mg/ml) dramatically diminished the lipid fluidity of the membrane, as estimated by electron spin resonance (ESR) spectrometry, and the ESR study revealed increased restriction of the molecular motion of the hydrophobic core in the treated membrane. These results suggest a drastic alteration of the erythrocyte membrane structure. The amount of drugs which penetrated into the treated erythrocytes increased markedly with increasing phenylhydrazine concentration, suggesting enhanced membrane permeability and facilitated localization of the drugs in the hydrophobic regions due to the structural changes and partial disturbance of the lipid organization.     

Effect of europium(III) chloride on the aggregation behavior of sodium dodecyl sulfate

The effect of EuCl3 on the aggregation processes of sodium dodecyl sulfate was investigated. Electrical conductivity data, combined with Eu(III) luminescence measurements, suggest that the formation of micelles involving EuCl3 and SDS occurs at low SDS concentration; the formation of these mixed aggregates was also monitored by light scattering, which indicates that the addition of EuCl3 to SDS concentration at values below the critical micelle concentration of the pure surfactant results in a much higher light scattering than that found just with SDS micelles. It was also found that the Eu(III)/DS- complexes are formed with a binding ratio which varies between 20 and 4, depending on the initial concentration of Eu(III). As the concentration increases, turbidity occurs initially, but solutions become clear subsequently. In contrast to the behavior of SDS in the presence of aluminum(III), no flocculation was observed. From the analysis of electrical conductivity data and comparison with other systems, it is suggested that growth of aggregates happens, probably with formation of nonspherical systems. At the highest concentrations these may involve just Eu(III) and DS- ions. The effect of temperature on the SDS micellization process was studied. The calculated free energy of SDS micellization is not dependent on the initial EuCl3 but is dependent on the final balance between the presence of counterions in solution (ionic strength) and the temperature.     

A novel LIF-CE method for the separation of hyaluronan- and chondroitin sulfate-derived disaccharides: Application to structural and quantitative analyses of human plasma low- and high-charged chondroitin sulfate isomers

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.     

Redox reactions of nitrite ions on the surface of colloidal magnetite particles coated with chondroitin sulfate

Colloidal magnetite particles coated with chondroitin sulfate (abbreviated as Fe/CS) were prepared under conditions of varying Fe(II) fractions at a fixed Fe concentration and a given concentration of CS. The average size of the magnetite core region was estimated as 7 nm from transmittance electron microscopy measurements, while the size of the Fe/CS particles ranged 155–175 nm, as estimated using Rayleigh scattering measurements by reference to a control size derived from the dynamic light scattering. The reaction of various Fe/CS with NO⁻ ₂ in aqueous solutions was determined by fluorometry using 2,3-diaminonaphthalene as a probe and by gas chromatography–mass spectrometry. The concentrations of NO⁻ ₂ in the reaction mixtures decreased in the presence of Fe/CS to a greater extent under Ar compared with aerobic conditions. The reactivity of Fe/CS toward NO⁻ ₂ under aerobic conditions increased with decreasing the size of Fe/CS particles or with increasing content of Fe(III) in the Fe/CS solutions, but was independent of the Fe(II) fraction in the preparation process. While CS molecules had no influence on the NO⁻ ₂ decomposition, those coated with the magnetite core may prevent the diffusion of NO⁻ ₂ to be adsorbed on the core surfaces. NO⁻ ₂ was concluded to undergo redox reactions with Fe(II) and Fe(III) located on the core surface of magnetite crystalline structures of Fe/CS. Received: 12 April 2000/Accepted: 9 August 2000