Anna Wisniewska

EFFECTS OF POLAR CAROTENOIDS ON THE SHAPE OF THE HYDROPHOBIC BARRIER OF PHOSPHOLIPID BILAYERS

The value of Az (z-component of the hyperfine interaction tensor) obtained directly from X-band EPR spectra of stearic acid spin labels and tempocholine dipalmitoylphosphatidic acid ester in frozen suspension of phosphatidylcholine (PC) membranes has been used as a hydrophobicity parameter. Using probes with the nitroxide moiety at various depths in the membrane, the shape of the hydrophobic barrier, which is determined by the extent of water penetration into the membrane, has been estimated. Incorporation of 10 mol% polar carotenoids, zeaxanthin, violaxanthin, or lutein into the saturated PC bilayer significantly increases the hydrophobicity of the membrane interior but decreases hydrophobicity (increases water penetration) in the polar headgroup region. Hydrophobicity at the membrane center increases from the level of propanolpentanol, which have dielectric constants of 10-20, to the level of dipropylamine, with a dielectric constant close to 3. Longer alkyl chains decrease the effect of polar carotenoids in the polar headgroup region, but not in the central hydrophobic region. In an unsaturated egg yolk PC membrane, polar carotenoids were found to increase the hydrophobicity of the membrane interior to a higher level than in saturated PC membranes. At the membrane center hydrophobicity reaches the level close to pure hexane (epsilon approximately 2). The above results were confirmed by studying accessibility of Fe(CN)6(3-) ion dissolved in water into dimyristoyl-PC-lutein membranes at 30 degrees C. Obtained hydrophobicity profiles correlate well with permeability data for water in the literature.

Location of macular xanthophylls in the most vulnerable regions of photoreceptor outer-segment membranes

Lutein and zeaxanthin are two dietary carotenoids that compose the macular pigment of the primate retina. Another carotenoid, meso-zeaxanthin, is formed from lutein in the retina. A membrane location is one possible site where these dipolar, terminally dihydroxylated carotenoids, named macular xanthophylls, are accumulated in the nerve fibers and photoreceptor outer segments. Macular xanthophylls are oriented perpendicular to the membrane surface, which ensures their high solubility, stability, and significant effects on membrane properties. It was recently shown that they are selectively accumulated in membrane domains that contain unsaturated phospholipids, and thus are located in the most vulnerable regions of the membrane. This location is ideal if they are to act as lipid antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular degeneration. In this mini-review, we examine published data on carotenoid-membrane interactions and present our hypothesis that the specific orientation and location of macular xanthophylls maximize their protective action in membranes of the eye retina.

Saturation-Recovery Electron Paramagnetic Resonance Discrimination by Oxygen Transport (DOT) Method for Characterizing Membrane Domains

The discrimination by oxygen transport (DOT) method is a dual-probe saturation-recovery electron paramagnetic resonance approach in which the observable parameter is the spin-lattice relaxation time (T1) of lipid spin labels, and the measured value is the bimolecular collision rate between molecular oxygen and the nitroxide moiety of spin labels. This method has proven to be extremely sensitive to changes in the local oxygen diffusion-concentration product (around the nitroxide moiety) because of the long T1 of lipid spin labels (1-10 micros) and also because molecular oxygen is a unique probe molecule. Molecular oxygen is paramagnetic, small, and has the appropriate level of hydrophobicity that allows it to partition into various supramolecular structures such as different membrane domains. When located in two different membrane domains, the spin label alone most often cannot differentiate between these domains, giving very similar (indistinguishable) conventional electron paramagnetic resonance spectra and similar T1 values. However, even small differences in lipid packing in these domains will affect oxygen partitioning and oxygen diffusion, which can be easily detected by observing the different T1s from spin labels in these two locations in the presence of molecular oxygen. The DOT method allows one not only to distinguish between the different domains, but also to obtain the value of the oxygen diffusion-concentration product in these domains, which is a useful physical characteristic of the organization of lipids in domains. Profiles of the oxygen diffusion-concentration product (the oxygen transport parameter) in coexisting domains can be obtained in situ without the need for the physical separation of the two domains. Furthermore, under optimal conditions, the exchange rate of spin-labeled molecules between the two domains could be measured.

The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method

Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid- and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (lo), liquid-disordered (ld), and solid-ordered (so) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the lo phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the ld phase, and ∼2 times smaller than in the pure ESM membrane in the so phase. The overall change in the oxygen transport parameter is as large as ∼20-fold. Conventional EPR spectra show that 5-SASL is maximally immobilized at the phase boundary between regions with coexisting ld and lo phases or so and lo phases and the region with a single lo phase. The obtained results allowed for the construction of a phase diagram for the ESM-cholesterol membrane.

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition.

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASLs) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASLs lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9- > 12- > 5- > 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8 degrees C and the cooperative unit to 1/5 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.

Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behaviour of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2′-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)3). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)3-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)3. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.

Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues.

Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.

EPR study of spermine interaction with multilamellar phosphatidylcholine liposomes

The interaction of spermine with egg-yolk phosphatidylcholine liposomes was investigated. The EPR spin labeling technique evidenced that spermine induces modifications of some membrane functions of biological interest like water permeability and is a possible modulator of diffusion processes for charged and polar molecules. The association constant for a hypothesized complex between spermine and the phosphate group of phosphatidylcholine was evaluated by enzymatic methods.