5-doxylstearic Acid Research Articles

Toxicity of terpenes on fibroblast cells compared to their hemolytic potential and increase in erythrocyte membrane fluidity

Terpenes are considered potent skin permeation enhancers with low toxicity. Electron paramagnetic resonance (EPR) spectroscopy of the spin label 5-doxyl stearic acid (5-DSA) was used to monitor the effect of sesquiterpene nerolidol and various monoterpenes on membrane fluidity in erythrocyte and fibroblast cells. In addition, the hemolytic levels and cytotoxic effects on cultured fibroblast cells were also measured to investigate possible relationships between the cellular irritation potentials of terpenes and the ability to modify membrane fluidity. All terpenes increased cell membrane fluidity with no significant differences between the monoterpenes, but the effect of sesquiterpene was significantly greater than that of the monoterpenes. The IC50 values for the terpenes in the cytotoxicity assay indicated that 1,8-cineole showed lower cytotoxicity and α-terpineol and nerolidol showed higher cytotoxicity. The correlation between the hemolytic effect and the IC50 values for fibroblast viability was low (R=0.61); however, in both tests, nerolidol was among the most aggressive of terpenes and 1,8-cineole was among the least aggressive. Obtaining information concerning the toxicity and potency of terpenes could aid in the design of topical formulations optimized to facilitate drug absorption for the treatment of many skin diseases.

Characterization of the Microenvironments of Alkylamidoamine-N-oxide Surfactant Aggregates by the EPR Spin Labeling Method

Spin labeling EPR methods were applied to characterize the local microviscosity and micropolarity of micellar aggregates formed by 3-(alkanoylamino)ethyldimethylamine-N-oxides (alkyl = nonyl, undecyl and tridecyl) in an aqueous environment in the temperature range 297–337 K. Four doxylstearic derivatives: 5-doxylstearate methyl ester (5-DSE), 16-doxylstearate methyl ester (16-DSE), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes to monitor mainly the rotational correlation time τ R and the isotropic hyperfine splitting constant A N. In the case of anisotropic spectra, the order parameter S was also determined. The selected probes were found to be sensitive to the microenvironments whereby it was possible to characterize in detail local changes in composition and micelle formation.

Vitex Agnus-Castus L. Essential Oil Increases Human Erythrocyte Membrane Fluidity

Vitex Agnus-CastusL. Essential Oil Increases Human Erythrocyte Membrane FluidityErythrocyte membrane fluidity is related to their rheologic behavior, the dynamic quality of erythrocytes, which is tempted in hypertension and atherosclerosis. An increased risk of these and other cardiovascular diseases occurs in ageing women. Menopause-related conditions are often treated with hormone replacement therapy that may increase the risk of malignancies.Vitex agnus-castusL. essential oil contains various organic compounds (monoterpenes, sesquiterpenes and terpenoids), and is increasingly used as an alternative therapy for menopausal symptoms. These components of the oil may be incorporated into cell membranes, thereby changing the membrane fluidity. The aim of this study was to determine the effects ofVitex agnus-castusessential oil on human erythrocyte membrane fluidity at graded depths. We used Electron Paramagnetic Resonance spectroscopy and fatty acid spin probes (5-doxyl stearic acid and 12-doxyl stearic acid), whose spectra depend on membrane fluidity. After treatment withVitex agnus-castusessential oil the erythrocytes had a significant (p=0.029) and reversible increase in membrane fluidity in the deeper hydrophobic membrane regions, with no change (p>0.05) in fluidity near the membrane's hydrophilic surface. These results document increased fluidity of the human erythrocyte membrane byVitex agnus-castusessential oil, and this action may be useful in patients with menopause-related hypertension and other cardiovascular conditions.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

EPR study of thylakoid membrane dynamics in mutants of the carotenoid biosynthesis pathway of Synechocystis sp. PCC6803.

EPR spectroscopy using 5-doxylstearic acid (5-SASL) and 16-doxylstearic acid (16-SASL) spin probes was used to study the fluidity of thylakoid membranes. These were isolated from wild type Synechocystis and from several mutants in genes encoding selected enzymes of the carotenoid biosynthesis pathway and/or acyl-lipid desaturases. Cyanobacteria were cultivated at 25°C and 35°C under different light regimes: photoautotrophically (PAG) and/or in light-activated heterotrophic conditions (LAHG). The relative fluidity of membranes was estimated from EPR spectra based on the empirical outermost splitting parameter in a temperature range from 15°C to 40°C. Our findings demonstrate that in native thylakoid membranes the elimination of xanthophylls decreased fluidity in the inner membrane region under optimal growth conditions (25°C) and increased it under sublethal heat stress (35°C). This indicated that the overall fluidity of native photosynthetic membranes in cyanobacteria may be influenced by the ratio of polar to non-polar carotenoid pools under different environmental conditions.

The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy.The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins.The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane.These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

Structural Behavior and Interactions of Dendrimer within Lyotropic Liquid Crystals, Monitored by EPR Spectroscopy and Rheology

Micro- and macrostructural behaviors of three different lyotropic liquid crystals (LLCs) loaded with a dendrimer, namely second generation poly(propylene imine) (PPI-G2), were studied by means of rheology and electron paramagnetic resonance (EPR). The three mesophases were L(α), Q(224), and H(II) composed of glycerol monooleate (GMO) and water-PPI-G2 solution (and d-α-tocopherol (vitamin E) in the case of H(II)). We characterized the impact of PPI-G2 interactions with the components of the host mesophases on their structural characteristics on different length scales. The incorporation of PPI-G2 within the L(α) and H(II) systems induced the formation of more elastic hexagonal systems with a "solidlike" behavior, while in the Q(224) system a different trend with a "liquidlike" behavior was observed. As a result, the dendrimer induced a remarkable change in both the structural and viscoelastic properties of the systems. Hence, the microenvironment in the interface region within the systems was monitored by computer-aided EPR using 5-doxylstearic acid (5-DSA) as a pH-dependent probe. The microviscosity (τ) and order (S) of systems were found to be sensitive to the PPI-G2 presence: when PPI-G2 concentration increased, τ and S increased in both the L(α) and Q(224) systems. In the H(II) systems two trends were observed, reflecting a decrease in τ and S up to 10 wt % PPI-G2 and subsequently their increase at higher dendrimer concentrations. It was assessed that PPI-G2 interacted strongly with the GMO hydroxyl groups in the L(α) phase, with the water molecules in the Q(224) systems. In the H(II) mesophase strong interactions with both the water and GMO hydroxyl molecules were detected.

Probing dendron structure and nanoscale self-assembly using computer-aided analysis of EPR spectra

The self-assembly of dendritic molecules is an effective way of generating functionalised nanoscale architectures. This study reports a computer-aided analysis of the EPR spectra of 5 doxyl-stearic acid (5DSA) and Cu(II) probes interacting with self-assembling dendrons in water. The dendrons investigated have previously been reported as potential gene delivery vehicles, and possess amine surface groups, different dendritic architectures based on ether-amide or ester branching, and hydrophobic groups at the focal point which can encourage self-assembly in aqueous solution. The parameters extracted from computation provide information about both the structure and dynamics in solution and the interacting ability of the dendrons to be used in gene therapy. The hydrophobic 5DSA probe is able to effectively probe the hydrophobic core of self-assembled dendron nanostructures. It reports on the polarity of its local environment and is most affected by dendrons with two cholesterol units at the focal point, partly affected by dendrons with cholesterol groups at their focal point, but is unaffected by dendrons with a simple phenyl group at the focal point. This reflects the different modes of self-assembly observed for these dendrons. When Cu(II) is used as an EPR probe of the branched environment, it was found that at pH7, much of the Cu(II) was ‘external’ to the dendritic structure, presumably due to protonation of the peripheral amine groups. On gradually increasing the Cu(II) loading, and using computer-aided analysis, it was possible to quantify the levels of ‘internal’ (dendron-bound) and ‘external’ Cu(II) and it was found that this was strongly dependent on the structure of the dendritic branching and the ability of the dendron to self-assemble, with self-assembling ester dendrons being best able to bind the Cu(II). It was also possible to propose the nature of the copper binding sites associated with the ‘internal’ signal as either Cu–NO3 and Cu–N2O2 distorted square-planar coordination sites. The self-assembling ester based dendrons which also contain 1,2,3-triazole units, had higher levels of ‘internal’ Cu(II) and showed the latter form of coordination, while the other dendrons, with lower levels of Cu(II) uptake showed the former. In summary, this paper demonstrates that two complementary EPR probes can be used to provide information about different regions of a self-assembled dendritic architecture.

Plasma lipid pattern and red cell membrane structure in β-thalassemia patients in Jakarta

Background: Over the last 10 years, we have investigated thalassemia patients in Jakarta to obtain a comprehensive picture of iron overload, oxidative stress, and cell damage. Methods: In blood samples from 15 transfusion-dependent patients (group T), 5 non-transfused patients (group N) and 10 controls (group C), plasma lipids and lipoproteins, lipid-soluble vitamin E, malondialdehyde (MDA) and thiol status were measured.  Isolated  eryhtrocyte  membranes  were  investigated  with  electron  paramagnetic  resonance  (EPR)  spectroscopy  using doxyl-stearic acid and maleimido-proxyl spin lables. Data were analyzed statistically with ANOVA. Results: Plasma triglycerides were higher and cholesterol levels were lower in thalassemic patients compared to controls. Vitamin  E,  group  C:  21.8  vs  T:  6.2  μmol/L)  and  reactive  thiols  (C:  144  vs.  T:  61  μmol/L)  were  considerably  lower  in  transfused patients, who exert clear signs of oxidative stress (MDA, C: 1.96 vs T: 9.2 μmol/L) and of tissue cell damage, i.e.,  high  transaminases  plasma  levels.  Non-transfused  thalassemia  patients  have  slight  signs  of  oxidative  stress,  but  no  signifi  cant  indication  of  cell  damage.  Erythrocyte  membrane  parameters  from  EPR  spectroscopy  differ  considerably  between all groups. In transfusion-dependent patients the structure of the erythrocyte membrane and the gradients of polarity and fl uidity are destroyed in lipid domains; binding capacity of protein thiols in the membrane is lower and immobilized. Conclusion: In  tranfusion-dependent  thalassemic  patients,  plasma  lipid  pattern  and  oxidative  stress  are  associated  with structural damage of isolated erythrocyte membranes as measured by EPR spectroscopy with lipid and protein-thiol spin labels. (Med J Indones 2011; 20:178-84)

Stratum Corneum Lipid of Hairless Mouse Investigated by EPR

Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation was utilized for examining the effect of diluted detergent-treated stratum corneum (SC) lipid structure. SC from the back of hairless mouse (HOS:HR-1) was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Aliphatic spin probe, 5-doxylstearic acid (5-DSA), was used to evaluate the SC ordering. The EPR spectrum of 5-DSA incorporated in the SC demonstrated a characteristic peak for the first strip. A slow-tumbling simulation for 5-DSA showed slight differences in ordering values (S 0) of the SC for the control and detergent-treated SC. The EPR spectra showed that the characteristic mobile component was eliminated. The values were 0.15 and 0.32, respectively. Thus, the EPR method along with the simulation analysis revealed the differences in ordering of the detergent-treated SC.

Structural Effects of Insulin-Loading into HII Mesophases Monitored by Electron Paramagnetic Resonance (EPR), Small Angle X-ray Spectroscopy (SAXS), and Attenuated Total Reflection Fourier Transform Spectroscopy (ATR-FTIR)

Insulin entrapment within a monoolein-based reverse hexagonal (H(II)) mesophase was investigated under temperature-dependent conditions at acidic (pH 3) and basic (pH 8) conditions. Studying the structure of the host H(II) system and the interactions of insulin under temperature-dependent conditions has great impact on the enhancement of its thermal stabilization and controlled release for the purposes of transdermal delivery. Small angle X-ray spectroscopy (SAXS) measurements show that pH variation and/or insulin entrapment preserve the hexagonal structure and do not influence the lattice parameter. Attenuated total reflection Fourier transform spectroscopy (ATR-FTIR) spectra indicate that, although insulin interacts with hydroxyl groups of GMO in the interface region, it is not affected by pH variations. Hence different microenvironments within the H(II) mesophase were monitored by a computer-aided electron paramagnetic resonance (EPR) analysis using 5-doxylstearic acid (5-DSA) as a pH-dependent probe. The microviscosity, micropolarity, order of systems, and distribution of the probes in different microenvironments were influenced by three factors: temperature, pH, and insulin solubilization. When the temperature is increased, microviscosity and order parameters decreased at both pH 3 and 8, presenting different decrease trends. It was found that, at pH 3, the protein perturbs the lipid structure while "pushing aside" the un-ionized 5-DSA probe to fit into the narrow water cylinders. At the interface region (pH 8), the probe was distributed in two differently structured environments that significantly modifies by increasing temperature. Insulin loading within the H(II) mesophase decreased the order and microviscosity of both the microenvironments and increased their micropolarity. Finally, the EPR analysis also provides information about the unfolding/denaturation of insulin within the channel at high temperatures.

Characterization of cephalexin loaded nonionic microemulsions

Water/propylene glycol/sucrose laurate/ethoxylated mono-di-glyceride/isopropyl myristate/peppermint oil U-type microemulsions were used to solubilize cephalexin. Microemulsion dilution and interfacial factors contributing to the cephalexin solubilization were evaluated. Cephalexin solubilization capacity increases with the increase in the aqueous phase volume fraction (φ) up to 0.4 then decreases. Electrical conductivity of drug loaded and drug free microemulsions increases with φ. The hydrodynamic radius measured by dynamic light scattering of the oil-in-water loaded microemulsions decreases with temperature. The microemulsions were characterized by the volumetric parameters, density, excess volume, ultrasonic velocity and isentropic compressibility. The microemulsion densities increase with φ up to 0.8 then decrease. The excess volume decreases with φ up to 0.8 then stabilizes. Ultrasonic velocities increase with the increase in φ while isentropic compressibility decreases. Analysis of the volumetric parameters enabled the characterization of structural transition along the microemulsion phase region. The presence of water-in-oil, bicontinuous and oil-in-water microemulsions, at aqueous phase volume fractions below 0.2, between 0.3 and 0.7 and above 0.8, respectively were found. Interfacial properties and dynamic structure of the monolayer for drug loaded and drug free microemulsions, were studied by electron paramagnetic resonance spectroscopy employing the nitroxide spin probe 5-doxylstearic acid. The rigidity of the interface was affected by the water content and also the presence of cephalexin.

Characterization of the Solution Structure of Human Serum Albumin Loaded with a Metal Porphyrin and Fatty Acids

The structure of human serum albumin loaded with a metal porphyrin and fatty acids in solution is characterized by orientation-selective double electron-electron resonance (DEER) spectroscopy. Human serum albumin, spin-labeled fatty acids, and Cu(II) protoporphyrin IX—a hemin analog—form a fully self-assembled system that allows obtaining distances and mutual orientations between the paramagnetic guest molecules. We report a simplified analysis for the orientation-selective DEER data which can be applied when the orientation selection of one spin in the spin pair dominates the orientation selection of the other spin. The dipolar spectra reveal a dominant distance of 3.85 nm and a dominant orientation of the spin-spin vectors between Cu(II) protoporphyrin IX and 16-doxyl stearic acid, the electron paramagnetic resonance reporter group of the latter being located near the entry points to the fatty acid binding sites. This observation is in contrast to crystallographic data that suggest an asymmetric distribution of the entry points in the protein and hence the occurrence of various distances. In conjunction with the findings of a recent DEER study, the obtained data are indicative of a symmetric distribution of the binding site entries on the protein's surface. The overall anisotropic shape of the protein is reflected by one spin-spin vector orientation dominating the DEER data.

Assessment of fluidity of different invasomes by electron spin resonance and differential scanning calorimetry

The aim of this study was to investigate the influence of membrane-softening components (terpenes/terpene mixtures, ethanol) on fluidity of phospholipid membranes in invasomes, which contain besides phosphatidylcholine and water, also ethanol and terpenes. Also mTHPC was incorporated into invasomes in order to study its molecular interaction with phospholipids in vesicular membranes. Fluidity of bilayers was investigated by electron spin resonance (ESR) using spin labels 5- and 16-doxyl stearic acid and by differential scanning calorimetry (DSC). Addition of 1% of a single terpene/terpene mixture led to significant fluidity increase around the C16 atom of phospholipid acyl chains comprising the vesicles. However, it was not possible to differentiate between the influences of single terpenes or terpene mixtures. Incorporation of mTHPC into the bilayer of vesicles decreased fluidity near the C16 atom of acyl chains, indicating its localization in the inner hydrophobic zone of bilayers. These results are in agreement with DSC measurements, which showed that terpenes increased fluidity of bilayers, while mTHPC decreased fluidity. Thus, invasomes represent vesicles with very high membrane fluidity. However, no direct correlation between fluidity of invasomes and their penetration enhancing ability was found, indicating that besides fluidity also other phenomena might be responsible for improved skin delivery of mTHPC.

Modification of the structure of plasmatic membranes of the liver by the action of α-tocopherol in vitro

The effect of the natural antioxidant alpha-tocopherol in a broad concentration range (10(-4) - 10(-25) M) on the viscosity characteristics and thermally induced structural transitions of a lipid bilayer of plasma membranes of murine hepatocytes in vitro has been studied. Changes in the rigidity of surface (approximately Abb) of the lipid bilayer were measured on a Bruker EMX EPR spectrometer (Germany) by the method of spin probes. Stable nitroxyl radicals of 5- and 16-doxylstearic acid, localized at different depth in the membrane served as spin probes. It was shown that the concentration dependence of the effect of alpha-tocopherol is linear and polymodal with three statistically significant increases in three ranges of its concentration: (1) in the range of traditional physiological concentrations 10(-4)-10(-9) M, (2) in the range of superlow doses 10(-9) - 10(-17) M, and (3) in the range of "imaginary" concentrations 10(-17) - 10(-25) M. The mechanisms of action of alpha-tocopherol in each of the three ranges are discussed. When studying the temperature dependences of viscous characteristics, a new thermally induced structural transition in the range of "physiological" temperatures 309-313 K for those alpha-tocopherol concentrations (including superlow ones) to which the maxima on the dose dependence curves at constant temperature of 293 K corresponded.

Elucidated lipid structures of various human stratum corneum investigated by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation is useful for defining stratum corneum (SC) lipid structure. The objectives of this investigation were to evaluate and elucidate the lipid structure of various human SC as a function of the depth. The SC from mid-volar forearms of human volunteers and a cadaver was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Spin probes were used to monitor SC ordering. EPR spectra were analyzed by an EPR slow-tumbling simulation. Keratin solution from human epidermis was mixed with 5-doxylstearic acid (5-DSA) aqueous solution. EPR analyses of 5-DSA and CHL probes showed immobilized and mobile spectra, respectively. The simulation for 5-DSA spectra showed differences in ordering values of the SC as a function of depth. EPR of keratin/5-DSA showed mobile spectral pattern. In addition, EPR of CHL showed a weak and a broad signal. The keratin/5-DSA results imply that the most likely location of 5-DSA probe is in SC lipid. CHL probe may not easily permeate the SC. The EPR results along with the simulation analysis provide elucidated SC lipid structure.

Nanostructuring of CyPLOS ( Cyclic Phosphate-Linked OligoSaccharides), novel saccharide-based synthetic ion transporters

Ionophores are an important class of synthetic molecules which mimic natural ion channels or carriers. Here we report the aggregation behavior in pseudo-physiological environment of three Cyclic Phosphate-Linked Oligosaccharides (CyPLOS) derivatives, synthetic ion transporters based on cyclic, phosphate-linked disaccharide skeleton differing for the nature of the tails (tetraethylene–TEG glycol and/or n-undecyl chains) attached to the C-2 and C-3 of the constitutive monosaccharides. Their aggregation behavior has been studied by a combined use of dynamic light scattering (DLS), electron paramagnetic resonance spectroscopy (EPR) and Small Angle Neutron Scattering (SANS). DLS measurements were performed to reveal the formation and size distribution of the CyPLOS aggregates. EPR measurements, by using 5-doxyl stearic acid (5-DSA) as spin-probe, showed that the aggregates are mainly due to the formation of double layers and allowed to analyze the local fluidity. Finally, SANS measurements allowed estimating the layer thickness of the double layers. Our results indicate that the three CyPLOS analogs show self-aggregation properties that depend on the different nature of the inserted tails.

Metalloporphyrin intercalation in liposome membranes: ESR study.

Liposomes characterized by membranes featuring diverse fluidity (liquid-crystalline and/or gel phase), prepared from egg yolk lecithin (EYL) and dipalmitoylphosphatidylcholine (DPPC), were doped with selected metalloporphyrins and the time-related structural and dynamic changes within the lipid double layer were investigated. Porphyrin complexes of Mg(II), Mn(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and the metal-free base were embedded into the particular liposome systems and tested for 350 h at 24°C using the electron spin resonance (ESR) spin probe technique. 5-DOXYL, 12-DOXYL, and 16-DOXYL stearic acid methyl ester spin labels were applied to explore the interior of the lipid bilayer. Only the 16-DOXYL spin probe detected evident structural changes inside the lipid system due to porphyrin intercalation. Fluidity of the lipid system and the type of the porphyrin complex introduced significantly affected the intermolecular interactions, which in certain cases may result in self-assembly of metalloporphyrin molecules within the liposome membrane, reflected in the presence of new lines in the relevant ESR spectra. The most pronounced time-related effects were demonstrated by the EYL liposomes (liquid-crystalline phase) when doped with Mg and Co porphyrins, whereas practically no spectral changes were revealed for the metal-free base and both the Ni and Zn dopants. ESR spectra of the porphyrin-doped gel phase of DPPC liposomes did not show any extra lines; however, they indicated the formation of a more rigid lipid medium. Electronic configuration of the porphyrin’s metal center appeared crucial to the degree of molecular reorganization within the phospholipid bilayer system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-010-0715-1) contains supplementary material, which is available to authorized users.

Stratum Corneum Lipid Structure Investigated by EPR Spin‐Probe Method: Application of Terpenes

Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation was utilized for defining stratum corneum (SC) lipid structure. SC from the back of hairless mouse (HOS:HR-1) was stripped consecutively from one to three or four times using a glass plate coated with a cyanoacrylate resin. Aliphatic spin probes, 5-doxylstearic acid (5-DSA) and 3β-doxyl-5α-cholestane (CHL), were used to evaluate the SC ordering. EPR spectrum of 5-DSA incorporated in the SC demonstrated a characteristic peak for the first strip. A slow-tumbling simulation for 5-DSA showed clear differences in EPR intensities as well as ordering values (S(0)) of the SC for control and terpenes treated SC. The α-terpineol enhanced the permeation of the single chain 5-DSA about three times more than that of the control. However, EPR spectra of CHL in the SC did not show a clear difference for each strip, except for the signal intensity. The results imply that CHL permeates into SC lipid differently from 5-DSA. The enhancement of the 5-DSA is more significant than that of CHL. Therefore, the present results can be useful for various drug administrations via the skin.

Interaction of Gum Arabic with Fatty Acid Studied Using Electron Paramagnetic Resonance

Electron paramagnetic resonance (EPR) is here used to study the interaction between gum arabic and a fatty acid. The EPR spectra of 5-doxyl stearic acid (5-DSA), a spin-labeled fatty acid analog, displayed increasingly anisotropic line features upon addition of gum arabic, indicating a strong immobilization of the nitroxyl moiety when the fatty acid is bound to gum arabic. To understand the nature of the interaction, EPR measurements were carried out at different pHs and using two fractions of gum arabic separated by hydrophobic interaction chromatography (HIC). 5-DSA bound favorably to the hydrophobic fraction, which contains mainly glycoprotein, and a small amount of high molecular weight arabinogalactan protein (AGP). Binding occurred to a less extent to the hydrophilic fraction, which contains essentially arabinogalactan (AG). Such a hydrophobic binding mechanism is further supported by a sharp drop in the binding when pH is raised above the pK(a) value of 5-DSA (approximately pH 5). This is because the ionization of carboxylic groups would lead to increased polarity and hydrophilicity of the fatty acid. A secondary effect involving the formation of ionic hydrogen bonds between carboxylic groups in fatty acid and lysine residues in gum arabic might also contribute. This is consistent with the reduction in binding ability when the pH was elevated above the pK(a) value of lysine residue (approximately pH 10). The biological significance of these findings is considered.