Cell Membrane Antioxidant Research Articles

Host–Guest Complexation of Amphiphilic Molecules at the Air–Water Interface Prevents Oxidation by Hydroxyl Radicals and Singlet Oxygen

AbstractThe oxidation of antioxidants by oxidizers imposes great challenges to both living organisms and the food industry. Here we show that the host–guest complexation of the carefully designed, positively charged, amphiphilic guanidinocalix[5]arene pentadodecyl ether (GC5A‐12C) and negatively charged oleic acid (OA), a well‐known cell membrane antioxidant, prevents the oxidation of the complex monolayers at the air–water interface from two potent oxidizers hydroxyl radicals (OH) and singlet delta oxygen (SDO). OH is generated from the gas phase and attacks from the top of the monolayer, while SDO is generated inside the monolayer and attacks amphiphiles from a lateral direction. Field‐induced droplet ionization mass spectrometry results have demonstrated that the host–guest complexation achieves steric shielding and prevents both types of oxidation as a result of the tight and “sleeved in” physical arrangement, rather than the chemical reactivity, of the complexes.

Vitamin E's Affinity for Polyunsaturated Phospholipids Studied by All-Atom MD Simulations

Vitamin E (α-tocopherol) is the principle lipid soluble antioxidant in cell membranes. Its purpose is to protect membrane lipids from oxidative damage. With all-atom (AA) MD simulations we address how affinity to various lipid species optimizes vitamin E's ability to protect polyunsaturated phospholipids that are most vulnerable to oxidation. Umbrella sampling simulations reveal that the binding energy of vitamin E simulations is greater for saturated SM (sphingomyelin) than polyunsaturated PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine). Preferential affinity for polyunsaturated phospholipids is not identified. The binding energy is reduced when cholesterol is added, however, suggesting vitamin E, like polyunsaturated phospholipids, is forced out of (raft-like) regions enriched in cholesterol into (non-raft) regions depleted in cholesterol. With large scale, microsecond-long simulations run on the Anton 2 supercomputer we determine where vitamin E prefers to locate in bilayers assembled with pre-formed PDPC and SM/cholesterol domains. The results of these studies will be presented.

A Role for Lipid-Lipid Interactions in Vitamin E's Function as a Membrane Antioxidant

Vitamin E (α-tocopherol) is the principle lipid soluble antioxidant in cell membranes. Its purpose is to protect membrane lipids from oxidative damage. Whether unequal affinity for different lipids optimizes the proximity of vitamin E to polyunsaturated phospholipids, the lipid species most susceptible to oxidation, is the question that we address with MD simulations on lipid bilayers. Our studies suggest cholesterol, ubiquitous in the cell membranes of animals, excludes vitamin E from saturated raft-like domains enriched in the sterol. Preferential affinity for polyunsaturated phospholipids is not indicated - vitamin E, like polyunsaturated phospholipids, is pushed towards non-raft regions depleted in cholesterol. The binding energy measured for vitamin E in umbrella sampling AA (all-atom) simulations is greater for SM (sphingomyelin) than PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine). Adding cholesterol to SM eliminates the differential in binding energy. CG (coarse-grained) simulations run on PDPC/SM/cholesterol mixtures in the presence of vitamin E indicate the vitamin locates at the boundary between SM-rich/sterol-rich (raft-like) and PDPC-rich/sterol-poor (non-raft) domains. CG simulations run on mixtures replacing PDPC by DDPC (1,2-didocosahexanoyl-sn-glycero-3-phosphocholine) will reveal details of the molecular interactions of vitamin E at the distinct boundary between raft-like and non-raft domains that forms with the di-polyunsaturated phospholipid. The results of these studies will be presented.

High-performance liquid chromatographic method for simultaneous determination of iloperidone and idebenone in spiked plasma

Background: Iloperidone (ILP) is a second-generation atypical antipsychotic agent and idebenone (IDB), a synthetic analogue of coenzyme Q10 is an important cell membrane antioxidant. No validated analytical method for simultaneous determination of ILP and IDB in rat plasma has been reported till date. Objective: To develop and validate a simple reversed phase high performance liquid chromatographymethodforsimultaneousdeterminationofiloperidoneandidebenoneinratplasma. Methods: A liquideliquid extraction method was used for deproteination of plasma samples using methanol as an extraction solvent. Chromatographic separations were done using isocratic conditions. Mobile phase containing acetonitrile and 0.025 M KH2PO4, pH 6 (60:40) at a flow rate of 1 mL/min was utilized for efficient separation. The UV detector was set at 277 nm. Risperidone was used as an internal standard. Results: The limits of detection (LOD) for iloperidone and idebenone were 10 and 20 ng/ml, while the limits of quantification (LOQ) were 30 and 35 ng/ml, respectively. The standard curves for iloperidone and idebenone in plasma were linear over the range of 0.05e20 mg/ ml, with the correlation coefficients of 0.9993 and 0.9985, respectively. All the validation parameters, such as accuracy, intra and inter-day precision were within the required limits. The samples were stable at � 80

Interaction of α-Tocopherol with a Polyunsaturated Lipid Studied by MD Simulations

Polyunsaturated phospholipids are essential components of neural membranes and their effect on membrane architecture is proposed to be the molecular origin of a myriad of health benefits. A downside of polyunsaturated phospholipids is that they are highly susceptible to oxidation due to the presence of multiple double bonds. α-Tocopherol is the most biologically active component in a family of phenolic compounds that comprise vitamin E, which is the major lipid soluble antioxidant in cell membranes. To investigate whether α-tocopherol preferentially interacts with polyunsaturated phospholipids to optimize protection against oxidation, we performed MD simulations on 1-stearoyl-2-docosahexaenoylphosphatiylcholine (SDPC, 18:0-22:6PC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) bilayers containing α-tocopherol. SDPC with a docosahexaenoyl sn-2 chain is polyunsaturated, while SOPC with an oleoyl sn-2 chain serves as a monounsaturated control. The simulations were run under constant pressure for 200 ns on a system that comprised 80 phospholipid molecules, 20 α-tocopherol molecules and 2165 water molecules. We discovered significant differences between the two systems. Notably, α-tocopherol produces a greater increase in order parameters for the stearoyl sn-1 chain of SDPC than SOPC, suggesting stronger interaction with the polyunsaturated phospholipid, and the flip-flop of α-tocopherol across the bilayer is much faster in SDPC than SOPC. We have further quantified the interaction of α-tocopherol with phospholipid by calculating the van der Waals interaction energy between α-tocopherol and the individual lipid chains (sn-1 and sn-2) in SDPC and SOPC. Solid state NMR, neutron scattering and complementary experiments are now underway to test the predictions from the MD simulations.

Effects of inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human fibroblasts.

Coenzyme Q10 (CoQ10) is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ10 biosynthesis. We observed a unimodal distribution of ROS production with CoQ10 deficiency: cells with <20% of CoQ10 and 50–70% of CoQ10 did not generate excess ROS while cells with 30–45% of CoQ10 showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ10 deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB), an analog of 4-hydroxybenzoate (4-HB) and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2) to induce a range of CoQ10 deficiencies. Our results support the concept that the degree of CoQ10 deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40–50% residual CoQ10 produces maximal oxidative stress and cell death.

Molecular Organization of Vitamin E in a Polyunsaturated Membrane

Vitamin E (α-tocopherol) is the major antioxidant in cell membranes. How when only present in very low concentration it can protect the comparatively large amount of polyunsaturated fatty acid (PUFA)-containing phospholipids that are particularly susceptible to oxidative attack is an unanswered question. Here we investigate the hypothesis that α-tocopherol laterally segregates into domains enriched in PUFA-containing phospholipids, thereby amplifying the concentration of the vitamin in the place where it is most needed. A combination of molecular dynamics (MD) simulations and solid state 2H NMR spectroscopy is employed to compare the molecular organization of α-tocopherol in 1,2-dipalmitoylphosphatidylcholine (DPPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and 1-stearoyl-2-docosahexaenoylphosphatiylcholine (SDPC) membranes. Our aim is to ascertain whether the highly disordered environment within polyunsaturated SDPC is more favorable to incorporation of α-tocopherol than the more ordered environment within saturated DPPC and monounsaturated SOPC.

Shortage of Lipid-radical Cycles in Membranes as a Possible Prime Cause of Energetic Failure in Aging and Alzheimer Disease

Polyunsaturated fatty acids (PUFA) and alpha-tocopherol (alpha-TOH) are the most oxygen-sensitive constituents of cells. alpha-TOH is a member of the vitamin E family that is considered the most important lipophilic antioxidant in cell membranes. Its importance is emphasized by the involvement of oxidative stress in injury to the central nervous system and neurodegenerative diseases. Currently, alpha-TOH transfer protein (TTP), is believed to play a significant role in maintaining the vitamin status but the presence of alpha-TOH in membranes is required but not sufficient to protect the membranes against lipid hydroperoxides (LOOH) formation. The lipid-radical theory presented in this review considers the role of two membrane factors--alpha-tocopherol and cytochrome b5; these factors secure the functioning of lipid-radical cycles and the participation of lipid-radical reactions in the key membrane processes. The prominent intermembrane reaction realized via a protein-lipid interaction, during which electron transport from cytochrome b5--located in the outer membrane--to peroxyl radical (LOO*)--located in inner membrane--causes reduction of the peroxyl radical: cyt.b5red + LOO* --> cyt.b5ox + LOO(-). This secures an interaction of alpha-TOH with other intermediate, LOO(- )excepting the LOOH formation. The discussion will be focused on the consequences of ineffective electron transfer to LOO* and excessive oxidative pathway of metabolism of the PUFA (LOO* --> LOOH). Assuming the operation of cytochrome b5/alpha-tocopherol-controlled lipid-radical cycles and considering the role of the cycles in membrane bioenergetics we arrive at a model for effective function of adenine nucleotide translocator and ATP synthesis in mitochondria. This paper summarizes our experimental evidence that the oxidative and non-oxidative pathways of metabolism of PUFA via their respective intermediates occur in the cells. While this fact is not widely appreciated it may be relevant to elucidation of new mechanisms of neurodegenerative diseases.

The reduction of nitroblue tetrazolium by red blood cells: a measure of red cell membrane antioxidant capacity and hemoglobin-membrane binding sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < 002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.

Nitric Oxide Inhibits Iron–Induced Lipid Peroxidation in HL-60 Cells

Nitric oxide (•NO) can protect cells against the detrimental effects of reactive oxygen species. Using low-density lipoprotein as well as model systems, it has been demonstrated that •NO can serve as a chain-breaking antioxidant to blunt lipid peroxidation. To test the hypothesis that •NO can serve as a chain-breaking antioxidant in cell membranes, we examined the effect of •NO on iron-induced lipid peroxidation in human leukemia cells. We exposed HL-60 cells to an oxidative stress (20 μM Fe2+) and monitored the consumption of oxygen as a measure of lipid peroxidation. Oxygen consumption was arrested by the addition of •NO as a saturated aqueous solution. The duration of inhibition of oxygen consumption by •NO was concentration-dependent in the 0.4–1.8 μM range. The inhibition ended upon depletion of •NO. The addition of •NO prior to initiation of peroxidation delayed the onset of peroxidation; the nearer in time it was before Fe2+ addition, the longer the inhibition. Depletion of cellular glutathione levels by d,l-buthionine-S,R-sulfoximine prior to Fe2+ addition resulted in a more rapid initial rate of oxygen depletion and a shorter time for the •NO-induced inhibition of oxygen consumption. Complementary studies of this iron-induced lipid peroxidation, using thiobarbituric acid reactive substances as a marker, also demonstrated the protective effects of •NO. This protection of cells against lipid peroxidation also manifested itself as a reduction in trypan blue uptake, an observation demonstrating the protective effects of •NO on membrane integrity. We conclude that •NO protects HL-60 human leukemia cells from lipid peroxidation and that this protection ameliorates the toxicity of the oxidation processes initiated by Fe2+ and dioxygen.

Vitamin A Inhibits Doxorubicin-Induced Membrane Lipid Peroxidation in Rat Tissues in Vivo

The antioxidant activity of vitamin A against lipid peroxidation induced by doxorubicin in rat tissues in vivo was investigated. A single ip injection of doxorubicin (30 mg/kg body wt) markedly raised the level of peroxidated lipids measured as TBARS and conjugated dienes in heart and brain membrane preparations. Other tissues, such as retina and liver, did not show any increase of lipid peroxides over control values. Pretreatment of rats with two daily subcutaneous injections of retinol palmitate (0.25 g/kg body wt), for 2 days, before injecting doxorubicin, inhibited peroxidation of heart and brain membrane lipids. The antioxidant action of vitamin A does not appear to be mediated by enhancement of antioxidant enzyme activities committed to detoxify oxygen radicals. Superoxide dismutase and catalase, measured in heart and brain cytosol, were not affected by the vitamin A treatment. On the contrary, a slight increase of catalase activity was observed in heart and brain cytosol from rats that had been treated with doxorubicin. Excess vitamin A may be localized in membranes. Appreciable increase of retinyl esters and retinol was measured in membrane preparations from rats that had been treated with vitamin A, with respect to control animals. Brain and heart membrane preparations from rats receiving vitamin A, assayed in vitro in the presence of an Fe3+ ascorbate induction system, showed a delay at the beginning of the lipid peroxidation and generated lesser amounts of TBARS, with respect to membranes from control rats. Thus, the increase of vitamin A within cell membranes results in an increased resistance of membrane lipids to peroxidation, both endogenously produced and induced in vitro. These results may be consistent with the hypothesis that vitamin A may act as a physiological antioxidant in cell membranes where it is localized.