Greta Pifat

The effects of essential oils and aqueous tea infusions of oregano (Origanum vulgare L. spp. hirtum), thyme (Thymus vulgaris L.) and wild thyme (Thymus serpyllum L.) on the copper-induced oxidation of human low-density lipoproteins

In this study, the antioxidative capacity effect of essential oils and aqueous tea infusions obtained from oregano, thyme and wild thyme on the oxidation susceptibility of low-density lipoproteins (LDL) has been studied. The results indicate a dose-dependent protective effect of the tested essential oils and aqueous tea infusions on the copper-induced LDL oxidation. The protective effect of essential oils is assigned to the presence of phenolic monoterpenes, thymol and carvacrol, which are identified as the dominant compounds in these essential oils. The strong protective effect of aqueous tea infusions is proposed to be the consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids (quercetin, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin), with the most pronounced effect in the case of oregano. These findings may have implications for the effect of these compounds on LDL in vivo.

Proton conductivity in the solid hydrated haemoglobin

Abstract 1. 1. Solid state electrolysis experiments were used to study the interaction of water vapour with bovine methaemoglobin. 2. 2. With 9.17% water adsorbed, which is equivalent to about one and half times the monolayer amount, there is no proton conduction, presumably because of lack of continuous hydrogen-bond chains. 3. 3. The existence of a hydration shell with such chains is indicated by the preponderance of proton conduction at a coverage corresponding to three monolayers. 4. 4. The measured energy of activation, 0.7 eV, is of the order encountered in protonic semiconductors. 5. 5. The dependence of the dielectric constant on hydration is also in agreement with this model. 6. 6. Permanent polarization after electrolysis of the 18% hydrated haemoglobin is ascribed to proton deficiency in the hydrogen bond network after electrolysis. 7. 7. At higher coverages, when “liquid” water is present between hydrated haemoglobin molecules, polarization decays in a few minutes. It is postulated that in this case a restoration of the initial state is accomplished by diffusion of protons from the “liquid” water into the hydration shell.

Nitroxide reduction with ascorbic acid in spin labeled human plasma LDL and VLDL

The LDL and VLDL were spin labeled with Tempo which partitions both in the aqueous and lipid phase. The ESR spectra were measured in the equilibrium state as well as during the reduction of the spin label with ascorbic acid. The kinetics of the concentration decay curves was parametrized with two exponentials. The theoretical simulation of the experimental spectra revealed a drastic linewidth narrowing in the VLDL samples exposed to the ascorbic acid. Since the transport properties of the specific monolayer are reflected in the observed reaction rates, the analysis of the fatty acid composition of phospholipids, triglycerides and cholesterol esters in LDL and VLDL was performed. It is concluded that different lipid packing at the surface of LDL and VLDL might be the consequence of different intermolecular forces between phospholipids and cholesterol. This finding was connected to the experimentally detected different reaction kinetics in LDL and VLDL as well as their different susceptibility to the ESR linebroadening effects during the nonequilibrium conditions of the spin label reduction with ascorbic acid.

The study of structural accessibility of free thiol groups in human low-density lipoproteins.

The experimental evidence for the apolipoprotein B100 (apoB) domain structuring in low-density lipoprotein (LDL) was investigated focusing on the accessibility of free thiol groups. Three different spectroscopic methods were combined with the biochemical perturbations of LDL particle. The spectrophotometric method was adapted for LDL and the exposure of free thiols was analyzed in the native LDL and LDL exposed to sequential denaturation. The results indicate that 24-h denaturation does not expose all free thiols in LDL. Using thiol-specific spin labeling and electron paramagnetic resonance spectroscopy (EPR), different populations of labeled thiols were resolved. The comparison of the EPR spectra of native LDL and LDL with selectively blocked thiol groups revealed significant difference in the respective hyperfine splittings. The phenomenon can arise due to different polarity and/or mobility of the nitroxides in the microenvironments of spin label binding sites of these two LDL samples. The results indicate that nine thiol groups in apoB are distributed in different domains of LDL: two are more exposed, two are buried deeply in the lipid matrix of the particle and the rest are located in hydrophobic parts of this extremely complex protein-lipid assembly. These observations provide experimental support for the emerging theoretical models of apoB.

The ESR characterization of molecular mobility in the lipid surface layer of human serum lipoproteins.

Three different nitroxides were used to probe either the head group (Tempil stearate) or acyl chain region (Spin labeled cholestane (ChSl) and methyl ester of 5 doxyl palmitate (MeFASL(10,3))) of human plasma low density lipoproteins (LDL) and very low density lipoproteins (VLDL). The ESR data were compared with the simulated spectra which assume rapid anisotropic motion of nitroxide. The results indicate that in the head group region of both LDL and VLDL only the slowing down of the rotational motion occurred when temperature was lowered and the whole region showed up as a unique compartment. On the other hand, the acyl chain region, probed with MeFASL(10,3), behaved as one compartment at physiological temperatures, while at lower temperatures coexistence of fluid and immobilized components were observed. The ESR spectra of lipoproteins labeled with Cholestane showed even higher sensitivity to the mobility constraints. Here, the LDL spectra revealed a drastic immobilization of ChSl axial rotation already at physiological temperatures. The results of these experiments were discussed in terms of core phase transition and/or lipid-protein interactions.

Surface-core correlations in serum lipoproteins

It has been found that the capacity of lipoproteins for binding Mn(II) ions is dependent on the arrangement of the Iipoprotein core. A change in the molecular organization of the LDL core (thermotropic transition) is associated with the change on the lipoprotein surface. In HDL there is no thermotropic transition and no abrupt change of the binding capacity.. The observed surface-core correlation might be an important link in understanding how dietary fat influences the lipoprotein metabolism.

EPR Investigation of Cell Membrane Fluidity upon External Oxidative Stimulus

The perturbation of the physical state of cell membranes triggered by an external oxidative stimulus has been studied with sperm cells which were chosen as a model system. Electron paramagnetic resonance (EPR) spectroscopy was applied and three different nitroxides bearing a paramagnetic group on the 5th, 7th and 13th carbon of the stearic acid acyl chain were used to probe different regions of the membrane. The theoretical simulations of the experimental spectra indicate distinct domains in the intact cell plasma membrane where local steric constraints impose different degrees of motional averaging experienced by the reporter group. Upon the external oxidative stimulus the spectral changes were proportional to the accumulated oxidation products and were detected only for the reporter group residing close to the lipid-water interface. The EPR spectra reveal that the motion of the reporter group has slowed down in the oxidized cells and that the oxidatively modified membrane shows up as a more rigid structure as compared with the native state. The results also indicate that the oxidation-induced spectral changes are supressed in the presence of gangliosides.

Causal relationship between the transitions in the core and the surface in porcine low-density lipoproteins

Two independent parameters, two characteristic temperatures, one indicating the change in the molecular organization of the core, Tc, and the other in the surface layer, Ts, were measured for a number of natural and triglyceride-enriched porcine low-density lipoprotein (LDL1 (buoyant density 1.020--1.063 g/ml) and LDL2 (buoyant density 1.063--1.080 g/ml) samples. Tc was determined by differential scanning calorimetry (DSC), whereas Ts was measured by Mn(II) binding to the lipoprotein surface followed by electron spin resonance (ESR) spectroscopy. A significant causal relationship between Tc and Ts in both LDL subfractions demonstrates the surface-core interaction in LDL. The significance of that interaction is emphasized as a possible link in the chain diet----lipoprotein changes----atherosclerosis.

Effect of gangliosides on the copper-induced oxidation of human low-density lipoproteins

The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.

An ESR study of the effect of an electrostatic field on binding of divalent cations to the surface of serum low-density lipoproteins

The ESR technique has been used to study binding of Mn(II) ions to low-density lipoprotein (LDL) in solutions of various electrolyte ionic strengths. A model of the binding has been proposed which describes all the observations in electrolytes of ten different concentrations in terms of two types of binding sites and two corresponding sets of intrinsic binding parameters (n1 = 8, Kd1 = 1.31 X 10(-3) mol X l-1 and n2 = 170, Kd2 = 5.71 X 10(-2) mol X l-1). These parameters, together with the values of the potential (phi 0) responsible for binding of the ions to specific charged sites on the surface, reproduce the observed binding curves well in all the systems studied. The phi 0 values are obtained as an appropriate solution of the Poisson-Boltzmann equation.