Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: Cholesterol product analysis as a probe of reaction mechanism

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [¹⁴C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5–1.0 mM ascorbate (AH⁻). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5α-hydroperoxide (5α-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH⁻/Fe(III) resulted in an almost-total disappearance of 5α-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7α-/7β-hydroperoxides (7α-/7β-OOH) and their respective alcohols (7α-/7β-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radical, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process. Presented at the symposium “Free Radicals Antioxidants, Skin Cancer and Related Diseases” at the 78th AOCS Annual Meeting in New Orleans, LA, May 1987.     

Singlet oxygen in copper-catalyzed lipid peroxidation in erythrocyte membranes

Lipid hydroperoxide was generated in human erythrocyte membranes by irradiation with near ultraviolet (UV) light in the presence of a photosensitizer, hematoporphyrin, but no production of 2-thiobarbituric acid-reactive materials (malonaldehyde and its precursors) was detected. Incubation of the irradiated membranes with CuSO₄ led to increased levels of hydroperoxide and formation of malonaldehyde. Hydroperoxides were essential for initiating the Cu(II)-catalyzed peroxidation as no significant activity was observed with nonirradiated membranes and Cu(II) unless an organic peroxide, either t-butyl hydroperoxide or cumene hydroperoxide, was added. Catalytic activity was also found with Fe(II), but not with other metal ions tested. The peroxidation catalyzed with Cu(II) was partially inhibited by several singlet oxygen quenchers but was not affected by superoxide dismutase, catalase or OH^• radical scavengers. The possible involvement of singlet oxygen in the Cu(II)-catalyzed peroxidation reaction was further supported by a 3-fold enhancement of malonaldehyde production in D₂O.     

Copper(II)-catalyzed lipid peroxidation in liposomes and erythrocyte membranes

Cu⁺⁺ was uniquely capable of catalyzing the peroxidation of rat erythrocyte membrane lipid in the presence of 10 mM H₂O₂, whereas several other transition metal ions were without significant effect. In contrast, peroxidation of soybean phospholipid liposomes could be catalyzed with decreasing efficiency by Co⁺⁺, Cu⁺⁺, Pb⁺⁺, or Cr⁺⁺⁺ also in the presence of H₂O₂. The effect of imidazole on Cu⁺⁺-catalyzed lipid peroxidation was stimulatory in liposomes and inhibitory in membrane preparations, whereas EDTA, histidine, citrate and alanine inhibited peroxidation in both systems. EDTA could stop the peroxidation after initiation, but catalase could not, indicating that Cu⁺⁺ alone was necessary for the propagation of the chain reaction. Competitive inhibition studies with various scavengers of hydroxyl radicals or singlet oxygen and the absence of significant reaction enhancement by D₂O indicated that neither of these reactive oxygen species was a major mediator in the Cu⁺⁺-H₂O₂ oxidative system. A copper-oxygen complex may be directly involved in the initiation of peroxidation. Normal erythrocyte membranes and phospholipid liposomes also differ in their sensitivities toward external oxidative stress. In the absence of H₂O₂, Cu⁺⁺ (0.2 mM) was capable of catalyzing lipid peroxidation in liposomes, aged erythrocyte membranes and membranes from vitamin-E-deficient rats; however, freshly prepared membranes from control rats and liposomes containing α-tocopherol required H₂O₂ greater than 2 mM for the catalytic effect of Cu⁺⁺ to be observed.     

Levels of Lipid Peroxidation in Human Plasma and Erythrocytes: Comparison between Fatty Acids and Cholesterol

Lipid peroxidation has gained renewed attention with increasing evidence showing its biological role in producing toxic compounds and cellular signaling mediators. The assessment of lipid peroxidation levels in vivo is difficult partly because lipids are oxidized by different oxidants by different mechanisms to give versatile types of products, which may undergo metabolism and secondary reactions. In the present study, total hydroxyoctadecadienoic acids (tHODE) and 7α- and 7β-hydroxycholesterol (t7-OHCh) from 44 healthy human subjects were assessed as biomarkers after reduction with sodium borohydride followed by saponification with potassium hydroxide comparing with the prevailing standard 8-isoprostaglandin F_2α (t8-iso-PGF_2α). The average concentrations of tHODE, total 8-isoprostaglandin F_2α (t8-iso-PGF_2α), t7α-OHCh, and t7β-OHCh were 203, 0.727, 87.1, and 156 nmol/l plasma and 1,917, 12.8, 1,372, and 3,854 nmol/l packed erythrocytes, respectively. The ratios of tHODE and t7-OHCh to the parent substrates were 194 and 3,519 μmol tHODE/mol linoleates and 40.9 and 686 μmol t7-OHCh/mol cholesterol in plasma and erythrocytes, respectively. It was found that (1) t7-OHCh in blood was unexpectedly high, as high as or even higher than tHODE, (2) the amounts of tHODE was more than 100 fold higher than t8-iso-PGF_2α (3) the level of lipid oxidation products in erythrocytes was higher than that in plasma, and (4) lipid peroxidation products level tended to increase while antioxidant level decrease with age. These products may be used as potential biomarker for assessment of lipid peroxidation and oxidative stress in vivo.     

Lipid peroxidation in chronic ethanol treated rats: In vitro uncoupling of peroxidation from reduced nicotine adenosine dinucleotide phosphate oxidation

Chronic ethanol treated rats were found to have enhanced ethanol metabolism and to metabolize ethanol in vivo in the presence of an inhibitor of alcohol dehydrogenase. In vitro studies of the hepatic microsomal system thought to the responsible for this activity showed it to be markedly induced. Lipid peroxidation also was enhanced in the ethanol treated animals. The lipid peroxidation was shown to be uncoupled from the microsomal nicotinamide adenine dinucleotide phosphate, reduced form, oxidase activity by a low concentration of azide.     

Modelling of organic liquid-phase decomposition reactions through gas-phase product analysis: Model systems and peracetic acid

A new method for modelling of liquid-phase decomposition reactions was developed. The method is based on rapid on-line mass spectrometric analysis of liberated products in gas phase. The experimental set-up and the generalized modelling principles were introduced. The approach was demonstrated with first and zero order as well as for complex decomposition kinetics (decomposition of peracetic acid). The interaction of decomposition kinetics and mass-transfer effects was illustrated with theoretical and real reaction conditions. The approach is generally applicable to any liquid-phase decomposition, provided that gaseous products can be quantitatively analyzed.     

Antioxidant Phenolics of Millet Control Lipid Peroxidation in Human LDL Cholesterol and Food Systems

Phenolic extracts from seven millet varieties, namely kodo, finger (Ravi), finger (local), proso, foxtail, little and pearl were evaluated for their inhibitory effects on lipid peroxidation in in-vitro copper-mediated human LDL cholesterol oxidation and several food model systems, namely cooked comminuted pork, stripped corn oil, and linoleic acid emulsion. The total phenolic content (TPC) and free radical scavenging activities were measured. The TPC ranged from 146 to 1156 μmol ferulic acid equiv (FAE)/g crude extract and the corresponding values based on defatted weight of grain ranged from 8.6 to 32.4 μmol FAE/g. At a final concentration of 0.05 mg/mL, millet extracts inhibited LDL cholesterol oxidation by 1–41%. All seven varieties exhibited effective inhibition of lipid oxidation in food systems used in this study and kodo millet exhibited superior inhibition of lipid peroxidation, similar to butylated hydroxyanisole at 200 ppm. Thus, millets may serve as a natural source of antioxidants in food applications and as a nutraceutical and functional food ingredient in health promotion and disease risk reduction.     

Peroxidation reactions in plant membranes: Effects of free fatty acids

Free fatty acids accumulate in plant membranes after exposure of plants to environmental stress, such as freezing and desiccation. Fatty acid accumulation has been linked to various biophysical changes and to the occurrence of lipid peroxidation, but the relationships appear complex and inconsistent. The interactions between oxygen free radicals, free fatty acids and lipid peroxidation in plant membranes were examined further by studying peroxidation reactions in a model membrane system composed of a complex mixture of plant phospholipids, including various free fatty acids. Multilamellar liposomes were treated with oxygen free radicals generated from iron ascorbate. Increased concentrations of free palmitic acid up to 10 mol% (fatty acid/phospholipid) reduced the production of aldehydes detected by the thiobarbituric acid assay, but enhanced the production of fluorescent products. By contrast, increased concentrations of free linolenic acid increased aldehyde production and reduced the formation of fluorescent products. The two free fatty acids both enhanced the susceptibility of phospholipids to degradation as shown by the reduced recovery of esterified polyunsaturated fatty acids (linoleic and linolenic). The free radical reactions with or without free fatty acid additions catalyzed the selective degradation of phospholipids in the order phosphatidylethanolamine > phosphatidylcholine > phosphatidylinositol > phosphatidylglycerol. Selective degradation of phospholipids is often observed after periods of environmental stress or during senescence of plants, and has been cited as evidence for the involvement of phospholipases in these degenerative processes. The results indicate that selectivity is not a criterion for eliminating the involvement of oxygen free radicals in these degenerative processes. Furthermore, the results suggest that modifications of lipid composition during a plant's acclimation to adverse environments may determine the types of free radical reactions that occur due to stress.     

Lipid peroxidation during n−3 fatty acid and vitamin E supplementation in humans

The purpose of this study was to investigate in healthy humans the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake, alone or in combination with dL-α-tocopherol acetate (vitamin E) supplements on lipid peroxidation. Eightly men were randomly assigned in a double-blind fashion to take daily for 6 wk either menhaden oil (6.26 g, n−3 fatty acids) or olive oil supplements with either vitamin E (900 IU) or its placebo. Antioxidant vitamins, phospholipid composition, malondialdehyde (MDA), and lipid peroxides were measured in the plasma at baseline and week 6. At the same time, breath alkane output was measured. Plasma α-tocopherol concentration increased in those receiving vitamin E (P<0.0001). In those supplemented with n−3 fatty acids, EPA and DHA increased in plasma phospholipids (P<0.0001) and plasma MDA and lipid peroxides increased (P<0.001 and P<0.05, respectively). Breath alkane output did not change significantly and vitamin E intake did not prevent the increase in lipid peroxidation during menhaden oil supplementation. The results demonstrate that supplementing the diet with n−3 fatty acids resulted in an increase in lipid peroxidation, as measured by plasma MDA release and lipid peroxide products, which was not suppressed by vitamin E supplementation.     

Lipid peroxidation in rat tissue homogenates: Interaction of iron and ascorbic acid as the normal catalytic mechanism

Iron and ascorbic acid appear to be the normal catalytic components responsible for the lipid peroxidation reaction in aerobically incubated rat tissue homogenates. The amounts of each present in the catalytically-active fractions of rat liver, brain, testis, and kidney are appropriate to explain the lipid peroxidation reaction measured. Utilization of ascorbic acid as part of the normal catalytic mechanism is indicated by the following: The catalytic activity of the tissue soluble phase occurs only in the small molecule fraction eluted from Sephadex, and ascorbic acid occurs only in this fraction; the extent of catalysis by the small molecule fractions of the soluble phases from several tissues is proportional to their ascorbic acid content; and pH effect on lipid peroxidation is the same for both soluble-phase and ascorbic acid catalysis. Utilization of iron as part of the normal catalytic mechanism is indicated by EDTA inhibition studies and by measurements of pH effects. Previous studies have demonstrated the lack of catalytic activity by cations other than iron for the lipid peroxidation reaction in homogenates. Lipid peroxidation is inhibited at high tissue concentration and the inhibition is due to components occurring in the large molecule fraction of the soluble phase.     

Lipid peroxidation in rat tissue slices: Effect of dietary vitamin E,corn oil-lard and menhaden oil

Rats were fed for 5 weeks either 10% (w/w) menhaden oil (MO) or a 10% corn oil-lard (COL) mixture (1∶1) in diets with ≤5 IU or ≤2 IU/kg vitamin E, respectively, or the same diets supplemented with d-α-tocopheryl succinate to a total of 35 and 180 IU vitamin E/kg, respectively. Slices of liver and heart from these rats were used to study lipid peroxidationin vitro. Thiobarbituric acid-reactive substances (TBARS) were measured in the medium after incubation of the slices at 37°C for 1 hr in the absence (uninduced) and presence of 0.5 mM tert-butyl hydroperoxide (induced). The release of TBARS from slices of heart and liver from rats fed either lipid decreased with increasing levels of dietary vitamin E. At the same level of dietary vitamin E, TBARS release was greater for slices of liver and heart from the MO-fed rats than from the COL-fed rats. Application of the TBARS data to a model simulating the experimental conditions showed a good correlation (r=0.95, p<0.001) between experimental and simulated values. Of the 16∶0–22∶6 fatty acids measured in liver from MO-fed rats, 15.4% was n−6 fatty acids and 29.9% was n−3 fatty acids; in liver from COL-fed rats, the respective values were 37.4% and 3.7%. Liver and kidney vitamin E levels were unaffected by the dietary lipid. This study demonstrated that a dietary fish oil increased the susceptibility of rat liver and heart toin vitro lipid peroxidation, and that vitamin E decreased TBARS in tissues from rats fed COL to lower levels than for tissues from rats fed MO. The results suggest that there might also be an increased requirement for dietary antioxidants by humans using fish oil supplements.     

钯膜及其在涉氢反应中的应用研究进展

综述了钯膜应用的研究进展，重点阐述了钯膜的透氢机理、制备方法、表征方法及在涉氢反应中的应用，总结了钯膜透氢性能的影响因素，指出了钯膜在分离和催化反应中面临的挑战，并对钯膜反应器的应用发展前景进行了展望。     

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior

Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100–200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela–Luc and H1299–Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74–98%) and low-serum circumstances (80–87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0–12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a “helper lipid-free” cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.     

NPC1L1 and SR-BI are Involved in Intestinal Cholesterol Absorption from Small-Size Lipid Donors

In the human intestinal content after a meal, cholesterol is dispersed in a complex mixture of emulsified droplets, vesicles, mixed micelles and precipitated material. The aim of this study was to determine the contribution of the main intestinal cholesterol transporters (NPC1L1, SR-BI) to the absorption processes, using different cholesterol-solubilizing donors. Cholesterol donors prepared with different taurocholate concentrations were added to an apical medium of differentiated TC7/Caco-2 cells. As the taurocholate concentrations increased, cholesterol donor size decreased (from 712 to 7 nm in diameter), which enhanced cholesterol absorption in a dose-dependent manner (38-fold). Two transport processes were observed: (1) absorption from large donors exhibited low-capacity transport with no noticeable transporter contribution; (2) efficient cholesterol absorption occurs from small lipid donors (相似文献   

Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Isoprostanes-biomarkers of lipid peroxidation: their utility in evaluating oxidative stress and analysis

Isoprostanes (IsoPs) are key biomarkers for investigating the role of free radical generation in the pathogenesis of human disorders. To solve IsoPs-related problems with regard to isoprostanes, analytical tools are required. This paper reviews the problems and trends in this field focusing on the methodology for assaying biomarkers in exhaled breath condensate (EBC) samples. A large amount of work has been done in the qualitative and quantitative analysis of IsoPs, but a standardized method has yet to emerge. The methodologies described differ, either in the sample preparation steps or in the detection techniques, or both. Requiring a number of chromatographic steps, the relevant extraction and purification procedures are often critical and time-consuming, and they lead to a substantial loss of target compounds. Recent data show that EBC is a promising non-invasive tool for the evaluation of different diseases. Two main analytical approaches have been adopted for IsoPs measurement: immunological methods and mass spectrometry. The methodologies for the extraction, purification and analysis of IsoPs in EBC samples are presented.     

Synergies between plant antioxidant blends in preventing peroxidation reactions in model and food oil systems

A study was conducted to investigate the oxidative behavior of various mixtures of rosemary, sage, and citric acid in a linoleic acid model system by oxygen consumption measurement and in a palm olein system by differential scanning calorimetry (DSC) analysis. Response surface methodology was used to optimize the use of the mixtures. Results showed that rosemary and sage were two important factors for the protective index (PI). The two antioxidants were highly significantly (P<0.001) in influencing PI values. There was a significant (P<0.01) synergistic effect between rosemary and sage on PI values. Citric acid was also found to be significant (P<0.05) for PI. With respect to onset time (T _o ), all three antioxidants were significant (P<0.05). However, no significant interaction among antioxidants was found for T _o . Mathematical models for both PI and T _o could be developed with confidence. The R ² values for PI and T _o were 0.992 and 0.926, respectively. A combination of 0.078% rosemary, 0.067% sage and 0.037% citric acid was the optimal combination for PI, whereas a combination of 0.068% rosemary, 0.075% sage, and 0.039% citric acid was required to reach the optimal T _o value.     

Bayesian analysis of color preferences: An application for product and product line design

When choosing which colors to offer in their product lines, firms often rely upon consumer preference models that do not account for the heterogeneity of their target market and do not consider the trade‐offs consumers are willing to make for different color options. For this research we used visual conjoint analysis to assess preference for backpack color and then modeled respondent utilities with a Bayesian hierarchal multinomial logit model. This provided counter intuitive results in which product line color options are not additive but each color changes depending on the number of options the firm is willing to offer and that colors which seem to dominate secondary preferences within a target market may not be the best colors to choose for product line expansion. © 2015 Wiley Periodicals, Inc. Col Res Appl, 41, 445–456, 2016