Radical-scavenging Activity of Estrogen and Estrogen-like Compounds Using the Induction Period Method

The radical-scavenging activity of estrogens (estrone, 2-hydroxyestradiol), estrogen-like compounds (diethylstilbestrol, DES; bisphenol A, BPA) and the monophenolic compound 2,6-di-t-butyl-4-methoxyphenol (BMP) was investigated using the method of measuring the induction period for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2′-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO) at 70°C using differential scanning calorimetry (DSC). The stoichiometric factor (n, number of free radicals trapped by one mole of antioxidant moiety) for the AIBN system declined in the order BMP (2.0), 2-hydroxyestradiol (2.0)> DES (1.3) > BPA (1.2) > estrone (0.9), whereas that for the BPO system declined in the order BMP (2.0) >DES (1.9), BPA (1.9) > estrone (1.3) > 2-hydroxyestradiol (0.7). The inhibition rate constant (k_inh × 10⁻³ M⁻¹s⁻¹) for the AIBN system declined in the order estrone (2.2) > BPA (2.0) > DES (1.9) > 2-hydroxyestradiol (1.2) > BMP (1.1), whereas that for the BPO system declined in the order 2-hydroxyestradiol (3.2) > estrone (1.4) > DES (1.2) > BPA (1.0) > BMP (0.9). The radical-scavenging activity for bioactive compounds such as estrogens should be evaluated using these two methods (the n and k_inh) to elucidate the mechanism of a particular reaction. The great difference of the n and k_inh for estrogens between the AIBN and BPO system suggested that their oxidation process is complex.     

Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer’s disease and are neurotoxic when oxidized ex vivo

Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer’s disease (AD). Recent studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein fraction (d<1.210 g/mL) was oxidized with 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator. Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis of neurodegeneration in AD.     

Effect of peroxyl radicals on lecithin/cholesterol acyltransferase activity in human plasma

The objective of this study was to determine whether subjecting human plasma to oxidant stress reduces the activity of lecithin/cholesterol acyltransferase (LCAT, EC 2.3.1.43). Plasma was incubated for 4h with 2.25–45 mM of 2,2′-azobis(2-amidinopropane)HCl (AAPH), a source of peroxyl radicals. A time- and concentration-dependent reduction of LCAT activity occurred, relative to control samples incubated in the absence of AAPH. Reduction of LCAT activity was disproportionate to elevation of thiobarbituric acid-reactive substances (TBARS) in the plasma. Added ascorbate was able to significantly prevent reduction of LCAT activity, but this effect was unrelated to blockage of TBARS formation by the antioxidant. The results suggest that LCAT activity can be down-modulated by oxidant stress, but not necessarily by lipid peroxidation.     

Ascorbate and phenolic antioxidant interactions in prevention of liposomal oxidation

Efficient prevention of membrane lipid peroxidation by vitamin E (α-tocopherol) may involve its regeneration by vitamin C (ascorbate). Conceivably, the efficacy of antioxidants designed as therapeutic agents could be enhanced if a similar regeneration were favorable; thus, a model membrane system was developed which allowed assessment of interaction of phenolic antioxidants with ascorbate and ascorbyl-6-palmitate. Ascorbate alone (50–200 μM) potentiated oxidation of soybean phosphatidylcholine liposomes by Fe²⁺/histidine-Fe³⁺, an effect which was temporally related to reduction of Fe³⁺ generated during oxidation. Addition of 200 μM ascorbate to α-tocopherol-containing liposomes (0.1 mol%) resulted in marked, synergistic protection. Accordingly, in the presence but not absence of ascorbate, α-tocopherol levels were maintained relatively constant during Fe²⁺/histidine-Fe³⁺ exposure. Probucol (4,4′-[(1-methylethylidine)bis(thio)]bis[2,6-bis(1,1-dimethylethyl)]phenol), and antioxidant which prevents oxidation of low density lipoproteins, and its analogues MDL 27,968 (4,4′-[(1-methylethylidene)bis(thio)]-bis[2,6-dimethyl]phenol) and MDL 28,881 (2,6-bis(1,1-dimethylethyl)-4-[(3,7,11-trimethyldodecyl)thio]phenol) prevented oxidation but exhibited no synergy with ascorbate. Ascorbyl-6-palmitate itself was an effective antioxidant but did not interact synergistically with any of the phenolic antioxidants. Differential scanning calorimetry revealed significant differences among the antioxidants in their effect on the liquid-crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC) liposomes. Both α-tocopherol and MDL 27,968 significantly reduced the phase transition temperature and the enthalpy of the transition. MDL 28,881 had no effect while probucol was intermediate. The potential for ascorbate or its analogues to interact with phenolic antioxidants to provide a more effective antioxidant system appears to be dictated by structural features and by the location of the antioxidants in the membrane.     

Comparison of <Emphasis Type="Italic">ex vivo</Emphasis> inhibitory effect between 2-hydroxyestradiol and its 17-sulfate on rat hepatic microsomal lipid peroxidation

Two endogenous antioxidants that are speculated to be defense substances against preeclampsia, 2-hydroxyestradiol (2-OH-E₂) and its 17-sulfate, 2-hydroxyestradiol 17-sulfate (2-OH-E₂-17-S), were administered to rats to compare their inhibitory effects on hepatic microsomal lipid peroxidation, and the lipid peroxides were determined in NADPH- and ascorbic acid-dependent systems. The two catechols showed a strong inhibitory effect on lipid peroxidation in both systems, and the effect was dose dependent. However, a large difference was observed in their inhibition patterns. After administration of 2-OH-E₂, the effect appeared immediately and decreased gradually with time. In contrast, the effect of 2-OH-E₂-17-S appeared some time after administration and persisted for a longer time. Both catechols also showed a striking difference in their dynamics. After administration, 2-OH-E₂ was detected in the blood together with its metabolites, 2-methoxyestradiol and 2-methoxyestrone, and they disappeared immediately. In contrast, 2-OH-E₂-17-S was present in the blood for a longer time together with its O-methylated product, 2-methoxyestradiol 17-sulfate, but disappeared from liver microsomes within 2 h after administration. The results imply no occurrence of a direct inhibition effect of 2-OH-E₂-17-S.     

The antioxidant properties of lycopene concentrate extracted from tomato paste

Lycopene concentrate (LC) containing 50 wt% lycopene was extracted from tomato paste. The antioxidant properties of LC were evaluated by means of chemiluminescence in four models. The four models were superoxide anions generated from pyrogallol autoxidation, hydroxyl radicals from Fenton reaction, singlet oxygens from OH⁻−NaClO−H₂O₂, and lipid peroxidation from 2,2′-azobis(2-amidinopropane)dihydrochloride-induced γ-linolenic acid. LC was an effective scavenger toward superoxide anions, hydroxyl radicals, and singlet oxygens, and also it could effectively reduce lipid peroxidation. The 50% efficient concentrations (EC₅₀) toward superoxide anions, hydroxyl radicals, lipid peroxidation and singlet oxygen were 0.75, 0.05, 0.1, and 1 mg/mL, respectively. In addition, changes of antioxidant behaviors with time were investigated. The time requirements of LC for effectively scavenging superoxide anions, hydroxyl radicals, and inhibiting lipid peroxidation were not higher than 6, 6, and 18 s, respectively.     

Chain-breaking fused heterocyclic antioxidants: Antioxidant activities of 9H-xanthene-2,7-diols and α-tocopherol upon liposomal membranes

We evaluated the antioxidant activities of 9H-xanthene-2,7-diols and α-tocopherol (α-Toc) upon the oxidation of soybean phosphatidylcholine liposomal membranes, induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azobis(2,4-dimethylvaleronitrile (AMVN). The stoichiometric factors of 9H-xanthene-2,7-diols, initiated with water-soluble AAPH and lipid-soluble AMVN, were 1.9–2.7 and 1.2–1.8-fold greater than those of α-Toc, respectively. The consumption profile of the antioxidant confirmed that 9H-xanthene-2,7-diol was completely consumed within the induction period (t _inh) and that the 9H-xanthene-2,7-diol oxidation product was formed. When all oxidation product was depleted, t _inh was terminated, and rapid oxidation occurred. These results suggested that the antioxidant activities of 9H-xanthene-2,7-diol depend not only on the initial hydrogen abstraction from 9H-xanthene-2,7-diol but also on a second hydrogen abstraction from the residual phenolic OH group of the oxidation product. Ascorbic acid (AsA) could not scavenge the radicals by itself in the lipid bilayer. However, when 9H-xanthene-2,7-diol was located in the lipid bilayer, the addition of AsA into the aqueous phase prolonged t _inh and reduced the rate of decay of 9H-xanthene-2,7-diol.     

Polyamine Oxidase Triggers H2O2-Mediated Spermidine Improved Oxidative Stress Tolerance of Tomato Seedlings Subjected to Saline-Alkaline Stress

Saline-alkaline stress is one of several major abiotic stresses in crop production. Exogenous spermidine (Spd) can effectively increase tomato saline-alkaline stress resistance by relieving membrane lipid peroxidation damage. However, the mechanism through which exogenous Spd pre-treatment triggers the tomato antioxidant system to resist saline-alkaline stress remains unclear. Whether H₂O₂ and polyamine oxidase (PAO) are involved in Spd-induced tomato saline-alkaline stress tolerance needs to be determined. Here, we investigated the role of PAO and H₂O₂ in exogenous Spd-induced tolerance of tomato to saline-alkaline stress. Results showed that Spd application increased the expression and activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and the ratio of reduced ascorbate (AsA) and glutathione (GSH) contents under saline-alkaline stress condition. Exogenous Spd treatment triggered endogenous H₂O₂ levels, SlPAO4 gene expression, as well as PAO activity under normal conditions. Inhibiting endogenous PAO activity by 1,8-diaminooctane (1,8-DO, an inhibitor of polyamine oxidase) significantly reduced H₂O₂ levels in the later stage. Moreover, inhibiting endogenous PAO or silencing the SlPAO4 gene increased the peroxidation damage of tomato leaves under saline-alkaline stress. These findings indicated that exogenous Spd treatment stimulated SlPAO4 gene expression and increased PAO activity, which mediated the elevation of H₂O₂ level under normal conditions. Consequently, the downstream antioxidant system was activated to eliminate excessive ROS accumulation and relieve membrane lipid peroxidation damage and growth inhibition under saline-alkaline stress. In conclusion, PAO triggered H₂O₂-mediated Spd-induced increase in the tolerance of tomato to saline-alkaline stress.     

Effects of membrane charges and hydroperoxides on Fe(II)-supported lipid peroxidation in liposomes

The processes in producing a lag phase in Fe²⁺-supported lipid peroxidation in liposomes were investigated. Incorporation of phosphatidylserine (PS) or dicetyl phosphate (DCP) into phosphatidylcholine [PC(A)] liposomes, which have arachidonic acid, produced a marked lag phase in Fe²⁺-supported peroxidation, where PS was more effective than DCP. Phosphatidylcholine dipalmitoyl [PC(DP)] with a net-neutral charge was still effective in producing a lag phase, though weak. Increasing concentrations of PS, DCP, and PC(DP) prolonged the lag period. Initially after adding Fe²⁺, slight oxygen consumption occurred in PC(A)/PS liposomes including hydroperoxides, followed by a lag phase. An increase in the hydroperoxide resulted in a shortening of the lag period. The initial events of Fe²⁺ oxidation accompanied by oxygen consumption were dependent on the hydroperoxide content, but significant changes in diene conjugation and hydroperoxide levels at this stage were not found. The molar ratios of both dis-appeared Fe²⁺ and consumed O₂ to preformed hydroperoxide in liposomes with or withouttert-butylhydroxytoluene were constant, regardless of the different amounts of lipid hydroper-oxides. The antioxidant completely inhibited the propagation of lipid peroxidation in the lipid phase, following a lag phase. In a model system containing 2,2′-azobis (2-amidinopropane) dihydrochloride, Fe²⁺ were consumed. We suggest that Fe²⁺ retained at a high level on membrane surfaces play a role in producing a lag phase following the terminating behavior of a sequence of free radical reactions initiated by hydroperoxide decompositin, probably by intercepting peroxyl radicals.     

Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method

Study of the role of hydroperoxides and lipid peroxidation in disease requires simple and sensitive methods for direct hydroperoxide measurement. We report on a technique for measuring hydroperoxide which relies upon the rapid hydroperoxide-mediated oxidation of Fe²⁺ under acidic conditions. Fe³⁺ forms a chromophore with xylenol orange which absorbs strongly at 560 nm, yielding an apparent E₅₆₀ (for H₂O₂, butyl hydroperoxide and cumene hydroperoxide) of 4.3×10⁴ M⁻¹ cm⁻¹. The assay was validated in a study of liposomal lipid peroxidation and shown to give results comparable with those obtained by an iodometric method or by measuring conjugated dienes. The assay involving thiobarbituric acid, by comparison, underestimates lipid peroxidation and does not measure hydroperoxideper se.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

The Allelochemical L-DOPA Increases Melanin Production and Reduces Reactive Oxygen Species in Soybean Roots

The non-protein amino acid, L-3,4-dihydroxyphenylalanine (L-DOPA), is the main allelochemical released from the roots of velvetbean and affects seed germination and root growth of several plant species. In the work presented here, we evaluated, in soybean roots, the effects of L-DOPA on the following: polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities; superoxide anion ( \textO₂^·- ) \left( {{\text{O}}_2^{{\bullet - }}} \right) , hydrogen peroxide (H₂O₂), and melanin contents; and lipid peroxidation. To this end, 3-day-old seedlings were cultivated in half-strength Hoagland’s solution (pH 6.0), with or without 0.1 to 1.0 mM L-DOPA in a growth chamber (at 25°C, with a light/dark photoperiod of 12/12 hr and a photon flux density of 280 μmol m⁻² s⁻¹) for 24 hr. The results showed that L-DOPA increased the PPO activity and, further, the melanin content. The activities of SOD and POD increased, but CAT activity decreased after the chemical exposure. The contents of reactive oxygen species (ROS), such as \textO₂^·- {\text{O}}_2^{{\bullet - }} and H₂O₂, and the levels of lipid peroxidation significantly decreased under all concentrations of L-DOPA tested. These results suggest that L-DOPA was absorbed by the soybean roots and metabolized to melanin. It was concluded that the reduction in the \textO₂^·- {\text{O}}_2^{{\bullet - }} and H₂O₂ contents and lipid peroxidation in soybean roots was due to the enhanced SOD and POD activities and thus a possible antioxidant role of L-DOPA.     

Tetrahydropyrrolization of Resveratrol and Other Stilbenes Improves Inhibitory Effects on DNA Oxidation

The inhibitory effect of resveratrol on DNA oxidation caused by 2,2′‐azobis(2‐amidinopropane hydrochloride) (AAPH) was found to be enhanced if the C=C bond in resveratrol was converted into tetrahydropyrrole by reaction with azomethine ylide (CH₂=N⁺(CH₃)CH₂^?). This encouraged us to explore whether the inhibitory activities of other stilbenes could also be increased by the same method. We found that the inhibitory effects of the tetrahydropyrrole derivatives on AAPH‐induced oxidation of DNA were higher than those of the corresponding stilbenes, because the tetrahydropyrrole motif can provide hydrogen atoms to be abstracted by radicals. Therefore, the tetrahydropyrrolization offered an advantage for enhancing the antioxidant effects of stilbenes. Notably, (CH₃)₃SiCH₂N(CH₃)CH₂OCH₃ (in the presence of CF₃COOH) and (CH₃)₃NO (in the presence of LiN(iPr)₂) can be used to generate azomethine ylide for the tetrahydropyrrolization of stilbenes containing electron‐withdrawing and ‐donating groups, respectively.     

Redox cycling of iron and lipid peroxidation

Mechanisms of iron-catalyzed lipid peroxidation depend on the presence or absence of preformed lipid hydroperoxides (LOOH). Preformed LOOH are decomposed by Fe(II) to highly reactive lipid alkoxyl radicals, which in turn promote the formation of new LOOH. However, in the absence of LOOH, both Fe²⁺ and Fe³⁺ must be available to initiate lipid peroxidation, with optimum activity occurring as the Fe²⁺/Fe³⁺ ratio approaches unity. The simultaneous availability of Fe²⁺ and Fe³⁺ can be achieved by oxidizing some Fe²⁺ with hydrogen peroxide or with chelators that favor autoxidation of Fe²⁺ by molecular oxygen. Alternatively, one can use Fe³⁺ and reductants like superoxide, ascorbate or thiols. In either case excess Fe²⁺ oxidation or Fe³⁺ reduction will inhibit lipid peroxidation by converting all the iron to the Fe³⁺ or Fe²⁺ form, respectively. Superoxide dismutase and catalase can affect lipid peroxidation by affecting iron reduction/oxidation and the formation of a (1∶1) Fe²⁺/Fe³⁺ ratio. Hydroxyl radical scavengers can also increase or decrease lipid peroxidation by affecting the redox cycling of iron. Based on a paper presented at the Symposium on Metals and Lipid Oxidation, held at the AOCS Annual Meeting in Baltimore, Maryland, April 1990.     

Antioxidative activity of 3,4-dihydroxyphenylacetic acid and caffeic acid in rat plasma

The purpose of the present paper is to study and compare in vitro the inhibitory effect of 3,4-dihydroxyphenylacetic acid (DOPAC) and caffeic acid (CA) on lipid peroxidation in rat plasma. Rat plasma was oxidized at 37°C by the radical initiators 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) or 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). The consumption of endogenous α-tocopherol (α-TOH) and the accumulation of conjugated diene hydroperoxides were measured by high-performance liquid chromatography and by ultraviolet spectroscopy, respectively. α-TOH was consumed at the same rate in the presence of 20 mM AAPH or 2 mM MeO-AMVN. DOPAC and CA suppressed the α-TOH consumption in a dose-dependent manner. A concentration of 50 μM of both phenolic acids was sufficient to induce a lag phase and to delay the rate of α-TOH consumption. The effect was more pronounced in rat plasma oxidation by AAPH than by MeO-AMVN. CA spared vitamin E more effectively than DOPAC in both oxidations. DOPAC and CA suppressed the formation of conjugated diene hydroperoxides. DOPAC and CA at concentration 50 μM suppressed α-TOH consumption during oxidation of soybean phosphatidylcholine (2.8 mM) multilamellar vesicles containing 15 μM α-TOH, in which the lipophilic initiator 2,2′-azobis (2,4-dimethylvaleronitrile) (6 mM) was incorporated. In conclusion, we demonstrated that DOPAC and CA in micromolar concentrations have antioxidant activity in rat plasma, a medium very close to the conditions in vivo, suggesting that supplementation with the phenolic acids will provide significant antioxidant protection.     

Quantification of Antioxidant Ability Against Lipid Peroxidation with an ‘Area Under Curve’ Approach

When oxygen is passed through a linoleic acid (LA) emulsion containing copper(II), primary (hydroperoxides) and secondary oxidation products (aldehydes and ketones) are formed, monitored by ferric thiocyanate (Fe(III)‐SCN) and thiobarbituric acid reactive substances (TBARS) colorimetry, respectively. As total antioxidant capacity (TAC) against lipid peroxidation was not quantified before, both methods were adapted to an ‘area under curve (AUC)’ approach. LA peroxidation followed pseudo‐first order kinetics in aerated emulsions. Absorbance changes as a function of incubation time exhibited sigmoidal curves, enabling the calculation of ‘area under curve’ (AUC) and net AUC = AUC_blank?AUC_sample, standard calibration curve as net AUC versus concentration, and trolox‐equivalent antioxidant capacity of the tested compounds. Garlic extract showed an antioxidative effect on hydroperoxide formation, but a prooxidative effect on TBARS. Although inhibition of lipid peroxidation was described qualitatively before, it was not evaluated quantitatively, e.g., the trolox‐equivalent antioxidant capacities (TEAC values) of antioxidants with respect to their inhibitive effect against lipid peroxidation were not calculated and compared. Additionally, real‐time monitoring of lipid oxidation products requires highly sophisticated but costly instrumental techniques, but no single oxidation product is a direct measure of lipid oxidation or its antioxidative prevention. The AUC approach is the first quantitative method measuring antioxidant protection against lipid oxidation, with a slightly different order of antioxidative effectiveness from reductive assays because of interfacial effects.     

The effect of beta blocking drugs on lipid peroxidation in rat heartin vitro

Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetolol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetolol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 μM FeSO₄, 5 μMt-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO₄ (liposomes) plus O₂. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetolol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation event at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.     

Peroxide dependent and independent lipid peroxidation: Site-specific mechanisms of initiation by chelated iron and inhibition by α-tocopherol

Peroxidation of linoleic acid (LA) was catalyzed by Fenton reagent (H₂O₂, and Fe²⁺) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecylsulfate (SDS) micelles. However, more hydroxyl radicals formedvia the Fenton reaction were trapped byN-t-butyl-α-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. Generation of linoleic acid alkoxy (LO) radicals by Fe²⁺ via reductive cleavage of linoleic acid hydroperoxide (LOOH) resulted in peroxidation of LA and formation of PBN-LO· adducts in SDS micelles, but not in TTAB micelles. This LOOH dependent lipid peroxidation could be catalyzed in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). LO radicals formed by the LOOH dependent Fenton reaction were also trapped by PBN at the surface of TTAB micelles in the presence of NTA, but not in its absence. The consumption of a spin probe, 16-(N-oxyl-4,4′-dimethyloxazolidin-2-yl)stearic acid (16-NS) during the LOOH dependent Fenton reaction in the presence of NTA was higher in TTAB micelles of LA than in those of lauric acid (LauA), although the rates and amounts of LO radicals formed in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same, and were lower than the rate of 16-NS in LA micelles. NTA-Fe²⁺ initiated peroxidation of LA in TTAB micelles without a lag time in the presence of LOOH, but after a lag period, peroxidation occurred without LOOH. α-Tocopherol inhibited peroxidation of LA catalyzed by Fenton reagent by scavenging OH radicals in TTAB micelles. In contrast, α-tocopherol enhanced free Fe²⁺ induced LOOH dependent lipid peroxidation through the regeneration of Fe²⁺ in SDS micelles. However, it inhibited NTA-Fe²⁺ induced LOOH dependent lipid peroxidation in TTAB micelles. The rate and amount of α-tocopherol oxidized by the Fe²⁺ induced, H₂O₂ dependent Fenton reaction were almost the same in TTAB micelles of LA and LauA. The oxidation of α-tocopherol by the NTA-Fe²⁺ induced, LOOH dependent Fenton reaction was greater and faster in LA micelles than in LauA micelles, although the rates of LO radical production in the two types of micelles were the same. During NTA-Fe²⁺ induced, LOOH dependent lipid peroxidation, α-tocopherol inhibited more effectively the consumption of 16-NS than 5-NS. The results are discussed in relation to the location of iron, the unsaturated bonding region of LA, the OOH group of LOOH, the radical trapping site of PBN, the spin sites of 5-NS and 16-NS, and the phenolic hydroxyl group of α-tocopherol in micelles with different charges. Based on a paper presented at the Symposium on Metals and Lipid Oxidation, held at the AOCS Annual Meeting in Baltimore, MD, April 1990.     

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO₂ for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increases were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO₂. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats was comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO₂ exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.     

Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate

The purpose of this study was to test the hypothesis that multiple antioxygenic nutrients provide increased protection against lipid peroxidative damage to rat liver. Rats were fed diets (i) deficient in vitamin E and selenium (Diet 1), (ii) supplemented with vitamin E and selenium (Diet 2), (iii) supplemented with (ii) and in addition trolox C,N-acetylcysteine, coenzyme Q₀, and (+)-catechin (Diet 3), or (iv) supplemented with (iii) and in addition β-carotene, ascorbic acid palmitate, canthaxanthin, and coenzyme Q₁₀ (Diet 4). Liver homogenates were obtained from three rats fed each of the diets for six weeks and were incubated at 37°C up to two hours with and without exogenous tertiary-butyl hydroperoxide (TBHP) or Cu²⁺. Lipid peroxidation was determined by measurement of thiobarbituric acid substances. Diets 2 and 3 significantly protected againstin vivo hepatic lipid peroxidation, and this protection was augmented by Diet 4. Diets 2, 3, and 4 were protective against mild oxidation induced by TBHP or Cu²⁺. During incubations with exogenous TBHP and Cu²⁺, there were only small differences between diets supplemented with antioxidants in inhibition of lipid peroxidation, indicating that diets supplemented with vitamin E and selenium (Diet 2) may have provided the maximal protection for liver. The possible mechanisms of protection provided by multiple antioxidants in diets were discussed. Protection by multiple antioxidants against lipid peroxidation may translate to prevention of peroxidative damage to human tissue, a factor in human disease.