Oxygen Diffusion-concentration Product Research Articles

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (PM) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the PM across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the PM of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.

Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ∼10°C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.

Discrimination and Characterization of Cholesterol Crystalline Domains using EPR Spin-Labeling: Application to Lens Lipid Membranes

Saturation-recovery EPR at X-band (9.4 GHz) and W-band (94 GHz) was used to discriminate cholesterol crystalline domains (CCDs) in model phospholipid membranes and membranes made of pigs lens lipids. We used dual-probe saturation-recovery EPR approach with cholesterol analogue spin labels, CSL or ASL, and oxygen (hydrophobic) or NiEDDA (polar) relaxation agents. The CCD was present in all of the phospholipid membranes when the cholesterol-to-phospholipid mixing ratio exceeded the cholesterol solubility threshold. Saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. However, in membranes made from nuclear lipids, two domains were detected and were assigned to the bulk phospholipid-cholesterol domain and the CCD, respectively. Profiles of the oxygen transport parameter (oxygen diffusion-concentration product) across these domains contain significant structural information. These profiles also allow calculating the oxygen permeability coefficient across domain. The evaluated upper limit of the oxygen permeability coefficient across the CCD at 37°C (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can form a barrier for oxygen transport in the lens nucleus. The new capabilities in measurements of the oxygen transport parameter at W-band are demonstrated and compared with results obtained at X-band. The loop-gap resonators used for W-band measurements have a sample volume of 30 nL, while the sample volume of the loop-gap resonator used for X-band is 3µL. These specifications ensure feasibility of experiments planned for samples with a limited sample volume. For example, the oxygen transport parameter profiles measured at X-band for lens lipid membranes were obtained for samples prepared from 50 - 100 eyes. Reliable profiles at W-band can be obtained based on samples prepared from one eye.

Three-dimensional Dynamic Structure Of Phospholipid Bilayers Saturated With Cholesterol

Membranes made of synthetic phospholipids, as well as total phospholipids extracted from the eye lenses of young and old animals and containing saturating amounts of cholesterol (close to or exceeding the cholesterol solubility threshold), were investigated using conventional and saturation-recovery EPR spin-labeling methods. Profiles of the order parameter and hydrophobicity were obtained from conventional EPR spectra. Profiles of the oxygen transport parameter (oxygen diffusion-concentration product) were obtained from saturation-recovery curves. All of these profiles provide unique information about the depth-dependent physical properties and the three-dimensional dynamic organization of the membrane. Additionally, saturation-recovery measurements allow discrimination of membrane domains because the collision rate of molecular oxygen with the nitroxide spin label may differ in these domains. All membranes saturated (but not oversaturated) with cholesterol are homogenous on the EPR timescale. When properties of the phospholipid-cholesterol membrane are monitored with phospholipid analogue spin labels (by measuring the alkyl chain order parameter), the membrane shows high rigidity that decreases gradually toward the membrane center. However, when membrane properties are measured by monitoring movement and/or concentration of small molecules like molecular oxygen or water, the monitored properties change abruptly between the C9 and C10 positions (depth to which the rigid cholesterol ring-structure is immersed), showing low membrane fluidity and hydrophobicity to the depth of the ninth carbon and high membrane fluidity and hydrophobicity in the membrane center. Based on these observations, it can be concluded that the bulk physical properties of membranes saturated with cholesterol (with a cholesterol-to-phospholipid mole ratio close to one) are mainly determined by the presence of the saturating amount of cholesterol and are practically independent on membrane phospholipid composition.

Effects of Wheat Antioxidants on Oxygen Diffusion−Concentration Products in Liposomes and mRNA Levels of HMG-CoA Reductase and Cholesterol 7α-Hydroxylase in Primary Rat Hepatocytes

Three wheat antioxidant fractions were investigated for their potential effects on oxygen diffusion-concentration products in liposomes prepared with egg yolk phosphatidycholine (yolk PC) and rat liver PC (liver PC), using the electron spin resonance (ESR) oximetry method with 2,2'-azobis(2-aminopropane) dihydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) as radical generators. Both water-soluble wheat antioxidant (WWA) and the second lipophilic antioxidant (LWA2) fractions were able to inhibit oxygen diffusion-concentration product induced by either AAPH or AMVN. The first lipophilic wheat antioxidant (LWA1) fraction only showed antioxidant activity in yolk PC liposomes with AAPH as the radical initiator but had pro-oxidant activity under other testing conditions. Both liposome composition and radical initiator altered the antioxidative properties of WWA, LWA1, and LWA2. WWA also showed the strongest DPPH(*) scavenging capacity on a per grain weight basis. HPLC analysis showed that WWA had a much higher level of total phenolic acids, which may partially explain their antioxidant properties. In addition, wheat antioxidants significantly down-regulated the mRNA of HMG-CoA reductase, the key enzyme for cholesterol biosynthesis, and up-regulated the mRNA of cholesterol 7alpha-hydroxylase (CYP7A1), the key enzyme for cholesterol metabolism, in primary rat hepatocytes. These data indicated the potential of wheat antioxidants in reducing the risk of atherosclerosis through multimechanisms.

Application of electron spin resonance (ESR) spectrometry in nutraceutical and food research

Electron spin resonance (ESR) spectroscopy is able to directly measure the chemical species with unpaired electrons and has been widely used in a number of research fields. This review focused on its application in nutraceutical and food research. Current status of ESR in free radical scavenging capacity estimation, food oxidative stability evaluation, Cu(2+) chelating capacity determination were summarized. Also discussed was the potential of ESR spin-label oximetry technique in examination of lipid peroxidation and oxygen diffusion-concentration products in liposomes, oxygen transport and depletion, and membrane structure and dynamic properties. In addition, ESR application in identifying and estimating irradiated foods including meat, fruits, vegetables, spices, cereal grains, and oil seeds was reviewed. Finally, the potential use of ESR technique in investigating microstructure change, phase transition and viscosity related properties during food formulation, processing, and storage was briefly mentioned, along with its potential in determination of radio-stability of food components. This review may provide some fundamental knowledge of ESR and its application in nutraceutical and food research.

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454–1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.

Effects of conjugated linoleic acid on oxygen diffusion-concentration product and depletion in membranes by using electron spin resonance spin-label oximetry.

The effect of conjugated linoleic acid (CLA) on the relation between structure and function of membranes is described in this paper. Electron spin resonance (ESR) spin-label oximetry was used in the present study to evaluate if oxygen transport and oxygen depletion were affected by incorporation of CLA instead of linoleic acid into membrane phospholipids. Specifically, 1-stearoyl-2-(9cis, 11 trans-octadecadienoyl)-phosphorylcholine (SCLAPC) was incorporated into soy plant phosphatidylcholine (soy PC) or egg yolk PC (EYPC) bilayers. The use of spin labels attached to different carbons along the fatty acid chain makes it possible to carry out structural and oximetric determinations with the same test sample. For example, the incorporation of 5 mol% SCLAPC increased the oxygen diffusion-concentration product in soy PC or EYPC liposomes at 37 degrees C, slightly decreased the ordering of the hydrocarbon chains at the C10 and C12 positions (in the region of the conjugated double bonds), and increased the rate of oxygen depletion from the aqueous medium. Similar results were not obtained by incorporating 5 mol% of 1-stearoyl-2-linoleoyl-PC (SLPC). In our model system, free-radical generation was initiated by extended incubation of the liposomes, by induction by 2,2'-azobis(2-amidinopropane)hydrochloride, or by ultraviolet irradiation of H2O2. The rate of consumption of molecular oxygen was studied by monitoring the oxygen concentration in the aqueous phases of the liposomes. The effect of 5 mol% SCLAPC in soy PC was significantly larger than 5 mol% SLPC in soy PC; the response patterns with soy PC and EYPC were similar. Furthermore, 5 mol% SCLAPC in 1-palmitoyl-2-linoleoyl-PC showed similar oxygen consumption to that observed with 5 mol% SCLAPC in EYPC. On the other hand, 5 mol% SCLAPC in synthetic PC membranes containing saturated or monounsaturated fatty acids showed low oxygen depletion rates. The perturbation of membrane structure and the increase of the relative oxygen diffusion-concentration products provided a potential mechanism by which CLA incorporated into membrane lipids could affect oxidative stress.

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.

Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study

The oxygen diffusion-concentration product was determined in phosphatidycholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25° C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474–4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.

Oxygen diffusion–concentration in phospholipidic model membranes. An electron spin resonance–saturation study

Fully hydrated liposomes of dipalmitoyl-phosphatidylcholine have been labelled with 5 (or 7, 10, 12, 16)-doxyl stearic acid at pH 6 and 8, and studied by the continuous-wave e.s.r.-saturation technique. The e.s.r. spectral magnitude depends on the hyperfrequency power P and on both T1 and T2 relaxation times. Saturation, i.e. the non-linearity of the spectral magnitude plotted vs.√P can be quantified by a P½ parameter (power at which the signal is half as great as it would be without saturation). If we assume T2 is weakly modified by spin exchange between the paramagnetic spin probe and oxygen in its triplet state, P½ is inversely proportional to T1 and becomes a sensitive parameter to appreciate the oxygen transport (oxygen diffusion–concentration product) inside the bilayers. According to the DPPC bilayer phase-transition diagrams, P½(oxygen diffusion–concentration) is related to the thermodynamic state of the membrane. This technique provides further information on a particular property of a radioprotective agent, cysteamine, which seems to inhibit spin–triplet exchange and hence maximizes T1(minimizes P½). Since radioprotective agents are known to act by scavenging radiation-induced free radicals and by inhibiting oxygen-dependent free radical processes, such a result may contribute to the elucidation of radioprotecting mechanisms.

The diffusion-concentration product of oxygen in lipid bilayers using the spin-label [formula omitted] method

A method is described to measure the oxygen diffusion-concentration product, D o [ O 2] , at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time T 1 of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to T 1 of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying T 1 are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in D o [ O 2] at the main phase transition was observed.