Małgorzata Komorowska

Influence of silybin on biophysical properties of phospholipid bilayers

AbstractAim:Silybin (silibinin) is major biologically active flavonolignan extracted from milk thistle (Sylibum marianum). Its biological activities include hepato-protection, anticancer properties, and antioxidant- and membrane-stabilizing functions. Although membranes are postulated to be one of the cellular targets for silybin, little is known about its interaction with phospholipid bilayers.Methods:In the present work, the interactions of silybin with phosphatidylcholine bilayers were studied in detail using fluorescence spectroscopy, microcalorimetry and electron spin resonance techniques.Results:The results showed that silybin interacted with the surface of lipid bilayers. It affected the generalized polarization of the fluorescent probe Prodan, while not influencing the more deeply located Laurdan. Silybin lowered the main phospholipid phase transition temperature as judged by microcalorimetry, and caused the immobilization of spin probe Tempo-palmitate located on the surface of membranes. The mobility of spin probes 5- and 16-doxyl stearic acid was not affected by silybin. Silybin-induced quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence indicated that some flavonoid molecules partitioned into the hydrophobic region of membranes, which did not change significantly the biophysical properties of the deeper membrane regions.Conclusion:Such a behavior of silybin in membranes is in accordance with its postulated biological functions and neglectable side effects of therapies using silybin.

The alterations of lipid bilayer fluidity induced by newly synthesized phenothiazine derivative

Using fluorescence spectroscopy, calorimetry and ESR the interactions of the phenothiazine derivative 2-trifluoromethyl-10-(4-[methylsulfonylamid]buthyl)-phenothiazine (FPhMS) with lipids were studied. Calorimetry showed biphasic effect of FPhMS on main phase transition of DPPC. At molar ratios up to 0.06 drug induced decrease of transition temperature and enthalpy, while at higher concentrations it caused subsequent increase of these parameters. For all concentrations studied we observed gradual broadening of transition peaks. Fluorescence polarization revealed that in FPhMS/lipid mixtures, order in bilayers is decreased in the gel state and increased in the liquid crystalline state. ESR experiment showed that at molar ratio of 0.06, FPhMS reduces the mobility of spin probes located in both polar and hydrophobic regions. Comparing observed effects with those reported for cholesterol/lipid mixtures, we conclude that at higher concentrations FPhMS presumably induces a new mode of bilayer packing. This structure is less co-operative than an unperturbed bilayer, but locally the mobility of lipid molecules is decreased.

FT-Raman spectroscopic study of human skin subjected to uniaxial stress.

Fourier Transform Raman Spectroscopy was used to investigate the molecular changes of structural proteins in human skin subjected to strain. In the Raman spectrum of unstrained skin, bands assigned mainly to collagen and elastin were observed at 1658 cm(-1) (amide I), 1271 and 1255 cm(-1) (amide III), and 935 and 817 cm(-1) (C-C stretching modes of the protein backbone). Moreover, bands characteristic for amino acids were observed at 1336 cm(-1) (desmosine), 1004 cm(-1) (phenylalanine), 919 and 856 cm(-1) (proline), and 877 cm(-1) (hydroxyproline). Positions and intensities of the listed Raman bands were analysed as a function of applied strain. A clear correlation between Raman wavenumbers and the level of mechanical stress was established. Wavenumbers of the analysed bands changed gradually with increasing strain. Distinct responses, depending on the sample cutting direction, i.e. longitudinal or perpendicular to the Langers lines, were noticed. It was concluded that elastin and non-helical domains of collagen are initially involved in the load transfer and triple helices of collagen are gradually joining this process. It was proved that Raman spectroscopy give insight into skin deformation micromechanics.

Application of FTIR-ATR Spectroscopy to Determine the Extent of Lipid Peroxidation in Plasma during Haemodialysis

During a haemodialysis (HD), because of the contact of blood with the surface of the dialyser, the immune system becomes activated and reactive oxygen species (ROS) are released into plasma. Particularly exposed to the ROS are lipids and proteins contained in plasma, which undergo peroxidation. The main breakdown product of oxidized lipids is the malondialdehyde (MDA). A common method for measuring the concentration of MDA is a thiobarbituric acid reactive substances (TBARS) method. Despite the formation of MDA in plasma during HD, its concentration decreases because it is removed from the blood in the dialyser. Therefore, this research proposes the Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) spectroscopy, which enables determination of primary peroxidation products. We examined the influence of the amount of hydrogen peroxide added to lipid suspension that was earlier extracted from plasma specimen on lipid peroxidation with use of TBARS and FTIR-ATR methods. Linear correlation between these methods was shown. The proposed method was effective during the evaluation of changes in the extent of lipid peroxidation in plasma during a haemodialysis in sheep. A measurement using the FTIR-ATR showed an increase in plasma lipid peroxidation after 15 and 240 minutes of treatment, while the TBARS concentration was respectively lower.

Erythrocyte response to near-infrared radiation

Abstract The effects of NIR (near-infrared radiation 700–2000 nm) on bovine erythrocytes in plasma was studied as a continuation of earlier studies. Cell shape was observed and the changes of ratio of hemolysis and electrokinetic potential measured as a function of irradiation time. After 10 min of irradiation, the shape of erythrocyte cells was mainly echinocytic. When these cells were incubated at 311 K for 24 h they regained their initial shape, but fresh erythrocytes that were irradiated for 30 min and aged in vitro did not. These phenomena are due to: (1) the absorption of NIR excitation by hemoglobin; the primary photochemical process being the photo-dissociation of oxyhemoglobin to deoxyhemoglobin. Resulting shape and ratio of hemolysis, structural changes and oxidative stress follow higher deoxyhemoglobin concentration. (2) The absorption of the NIR excitation by proteins, water and lipids. After NIR absorption the membrane surface dehydrates, leading to enhanced protonation and dissociation of hydrogen-bonded complexes. This in turn leads to a change in electrokinetic potential.

Near-infrared induced membrane surface electrostatic potential, fluorescent measurements.

The change of the electrostatic surface potential induced by near-infrared radiation was monitored by the fluorescence probe technique. Fluorescence intensity of 1-anilinonaphtalene-8-sulfate (ANS) was studied in the pH range 4.8-9.5 before and after exposition to NIR (700-2000 nm). The intensity of fluorescence changed (decreased after exposition on radiation) only at pH 7.4. The effect is due to decreasing concentration of ANS in liposome membrane after irradiation. The modified distribution of ANS in liposome membrane upon irradiation is attributed to the dehydration of membrane surface. Dehydratation diminishes the electrostatic surface potential about 36+/-15 mV.

Individual osmotic fragility distribution: a new parameter for determination of the osmotic properties of human red blood cells.

The aim of our experiments was to characterise and to validate the osmotic fragility test when applied to human blood samples with no significant alterations of osmotic fragility but with a differentiating shape of the haemolysis curve. All experiments were carried out on human erythrocytes taken from the Regional Centre of Blood Donation and Blood Therapy in Wrocław. The washed erythrocytes were exposed to near-infrared radiation (NIR) or ozonated, and the osmotic fragility test was applied. The osmotic fragility, calculated from the experimental haemolysis curve for the control and cells irradiated for 15 min, is the same within the empirical error. Calculation of the first derivative of the haemolysis curve allowed us to visualise the changes in osmotic fragility distribution after exposure to NIR. By contrast, significant changes both to the osmotic fragility value and the distribution of osmotic properties were observed after an erythrocytes ozonation procedure. Description of cell osmotic properties requires at least two parameters—the value of osmotic fragility and the slope of the haemolysis curve in the region where absorbance sharply increases due to cell haemolysis.

Hydration effects under near-infrared radiation

Abstract Changes in liposome membrane surface properties induced by near-infrared radiation (NIR) have been observed using two methods: spin labels and microscope observations. Multilamellar liposomes were prepared from egg yolk phosphatydylcholine (PC). Isotropic tumbling correlation time ( τ c ) for PC liposome membranes, calculated from EPR spectra, increased after irradiation (700–2000 nm). Both the Arrhenius plot temperature of discontinuity for the TEMPO-palmitate (TP) spin probe correlation time incorporated into the PC bilayer, and calculated activation energy of its mobility are shifted towards higher values after irradiation, but only at neutral pH. The ability of liposomes to agglomerate was modified considerably by irradiation when observed under optical microscope. All observed phenomena have been discussed as a result of NIR hydration effects.

Near-infrared-induced proton transfer studied by electron spin resonance

Abstract The effect of near-infrared (NIR) radiation (700–2000 nm) on water solution containing the nitroxide spin probe and selected amines has been studied. The concentration of free radical reversibly decreased upon irradiation. Explanation of this phenomenon has been based on the known properties of nitroxide radical in water. A reaction model for the process of reversible reduction of the nitroxide in water in the presence of amine and oxygen has been elaborated. The key reaction proposed for this process is NIR-induced decomposition of a complex between amine and nitroxide: R′NH 3 + ⋯ON ⋅ R.

Specific Applications of Vibrational Spectroscopy in Biomedical Engineering

Sylwia Olsztynska-Janus1, Marlena Gąsior-Glogowska1,4, Katarzyna Szymborska-Malek2, Boguslawa Czarnik-Matusewicz3 and Malgorzata Komorowska1,4 1Institute of Biomedical Engineering and Instrumentation, Wroclaw University of Technology, 2Institute of Physical and Theoretical Chemistry, Wroclaw University of Technology, 3Faculty of Chemistry, University of Wroclaw, 4Regional Specialist Hospital in Wroclaw, Research and Development Centre, Wroclaw, Poland