Comparison of the antioxidant action of the alcoholic extract of Rubia cordifolia with rubiadin.

The inhibition of FeSO4 induced lipid peroxidation in rat liver by alcoholic extract of Rubia cordifolia and by one of its constituent rubiadin (1, 3-dihydroxy-2-methyl anthraquinone) (pure form) has been compared. Both have been found to inhibit lipid peroxidation in a dose dependent manner. Whereas the former shows both oxidising and reducing properties with Fe2+ and Fe3+, the latter shows oxidising property only by converting Fe2+ to Fe3+. The former inhibits the oxidation of reduced glutathione while the latter does not.     

The interaction of Rubia cordifolia with iron redox status: a mechanistic aspect in free radical reactions.

The present publication investigates the antioxidant property and mechanistic aspect of alcoholic extract of R. cordifolia. The extract of R. cordifolia has shown significant inhibitory effect on FeSO4 induced lipid peroxidation. Study with iron redox status showed that R. cordifolia extract reduced or oxidixed; Fe3+ or Fe2+ respectively, in a dose dependent manner. Results with superoxide anion (O2-.) and hydroxyl radical (OH.), showed no radical scavenging activity. The alcoholic extract significantly maintains the reduced glutathione content both in time and dose dependent manner. It also reduced the rate of depletion of reduced glutathione (GSH) level in presence of ferrous sulphate (FeSO4) and cumene hydroperoxide (CHP). On the basis of these observations, it can be concluded that the antioxidant property of R. cordifolia is due to a direct interaction with iron.     

Free radical scavenging and metal chelation by Tinospora cordifolia,a possible role in radioprotection

Aqueous extract of T. cordifolia inhibited Fenton (FeSO4) reaction and radiation mediated 2-deoxyribose degradation in a dose dependent fashion with an IC50 value of 700 microg/ml for both Fenton and radiation mediated 2-DR degradation. Similarly, it showed a moderate but dose dependent inhibition of chemically generated superoxide anion at 500 microg/ml concentration and above with an IC50 value of 2000 microg/ml. Aqueous extract inhibited the formation of Fe2+-bipiridyl complex and formation of comet tail by chelating Fe2+ ions in a dose dependent manner with an IC50 value of 150 microg/ml for Fe2+-bipirydyl formation and maximally 200 microg/ml for comet tail formation, respectively. The extract inhibited ferrous sulphate mediated lipid peroxidation in a dose-dependent manner with an IC50 value of 1300 microg/ml and maximally (70%) at 2000 microg/ml. The results reveal that the direct and indirect antioxidant actions of T. cordifolia probably act in corroboration to manifest the overall radioprotective effects.     

3-O-beta-D-Galactopyranoside of quercetin as an active principle from high altitude Podophyllum hexandrum and evaluation of its radioprotective properties

The aqueous-ethanolic extract (AEE) of high altitude Podophyllum hexandrum has earlier been reported to render a radioprotective effect against lethal gamma radiation in in vitro model. AEE has also been reported to possess metal chelating and DNA protecting properties. The present study was undertaken to isolate and characterize the bioactive principle present in AEE and investigate its role in radiation protection. A novel molecule was found to be present in AEE and was assigned as 3-O-beta-D-galactoside of quercetin by acid hydrolysis, LC-MS, LC-APCI-MS/MS and 13C NMR spectra. Various biological activities were investigated at in vitro level. The antioxidant potential of AEE in lipid and aqueous phase was determined against numerous stresses. AEE was found to be significantly (p < 0.05) protective, i.e., against Fe2+ and Cu2+-induced linoleic acid degradation, respectively. Radiation-induced lipid oxidation studies revealed that AEE maximally works at a [lignan]/0.25 kGy ratio 400 (ratio of concentration of AEE divided by the radiation dose, i.e., 0.25 kGy) and no drug-induced lipid oxidation at all concentrations tested was found. In a time-dependent study, total antioxidant activity was maximally exhibited at 1 mg/ml. The site-specific and non-site-specific deoxyribose degradation assay exhibited a dose-dependant hydroxyl scavenging potential of AEE (0.05-500 microg/ml). The anti-lipid peroxidation ability of AEE against radiation (0.25 kGy)-induced lipid peroxidation was higher in case of neural tissue homogenate as compared to kidney homogenate [activity ratio: 0.039 (brain) < 0.24 (kidney)]. The protein protection study using bovine serum albumin was also done for two time intervals (2 h and 4 h) and significant (p < 0.05) protection was observed at 500 microg/ml (> 97%). This study implies that 3-O-beta-D-galactoside present in AEE renders radioprotection by protecting lipids, proteins in renal and neural model system against supra-lethal (0.25 kGy) gamma radiation.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Antioxidant properties of rimantadine in influenza virus infected mice and in some model systems

Influenza virus infection is associated with development of oxidative stress in lung and blood plasma, viz. increase of primary and secondary lipid peroxidation products. It was established that rimantadine treatment led to a decrease of the products of lipid peroxidation in tissues of mice experimentally infected with influenza virus A/Aichi/2/68 (H3N2). The effect is strongest in blood plasma (a decrease of about 50%) and weaker in the lung (about 20%). To elucidate the mechanism of this action of rimantadine, experiments were carried out with some model systems. The capability of rimantadine to scavenge superoxide radicals (scavenging properties) was studied in a system of xanthine-xanthine oxidase to generate superoxide. The amount of superoxide was measured spectrophotometrically by the NBT-test and chemiluminesce. Rimantadine does not show scavenging properties and its antioxidant effect observed in vivo, is not a result of its direct action on the processes of lipid peroxidation and/or interaction with antioxidant enzymes. The antioxidant properties of rimantadine were investigated by measurement of induced lipid peroxidation in a Fe2+ and (Fe2+ - EDTA) system with an egg liposomal suspension. Our findings with model systems do not prove an antioxidant or prooxidant effect of the drug on the processes of lipid peroxidation. Apparently, the observed antioxidant effect of rimantadine in vivo is not connected directly with free radical processes in the organism.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH(.)) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe3+ - Fe2+ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe2+) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 microg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH(.) scavenging, ABTS(.)+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe3+) reducing power by Fe3+-Fe2+ transformation, cupric ions (Cu2+) reducing ability by Cuprac method, and ferrous ions (Fe2+) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.     

Factors affecting the free radical scavenging behavior of chitosan sulfate

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, ^?OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl₄)-treated animals. It was observed that CCl₄-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.     

Antioxidant properties and protective effects of a standardized extract of Hypericum perforatum on hydrogen peroxide-induced oxidative damage in PC12 cells

Free radical scavenging and antioxidant activities of a standardized extract of Hypericum perforatum (SHP) were examined for inhibition of lipid peroxidation, for hydroxyl radical scavenging activity and interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). Concentrations between 1 and 50 microg/ml of SHP effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe2+/ascorbate or NADPH system. The results showed that SHP scavenged DPPH radical in a dose-dependent manner and also presented inhibitory effects on the activity of xanthine oxidase. In contrast, hydroxyl radical scavenging occurs at high doses. The protective effect of the standardized extract against H2O2-induced oxidative damage on the pheochromocytoma cell line PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays, caspase-3-enzyme activity and accumulation of reactive oxygen species [2',7'-dichlorofluorescin (DCF) assay]. Following 8-h cell exposure to H2O2 (300 microM), a marked reduction in cell survival was observed, which was significantly prevented by SHP (pre-incubated for 24 h) at 1-100 microg/ml. In a separate experiment, different concentrations of the standardized extract (0.1-100 microg/ml) also attenuated the increase in caspase-3 activity and suppressed the H2O2 -induced reactive oxygen species generation. Taken together, these results suggest that SHP shows relevant antioxidant activity both in vitro and in a cell system, by means of inhibiting free radical generation and lipid peroxidation.     

Effects of beta-carotene and alpha-tocopherol on radical-initiated peroxidation of microsomes.

Rat liver microsomal membranes were exposed to either beta-nicotinamide adenine dinucleotide phosphate (NADPH), adenosine 5'-diphosphate (ADP), and Fe+3 or to azocompounds, and the antioxidant activities of beta-carotene and alpha-tocopherol were studied. Lipid peroxidation was monitored either by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm or by hydroperoxide formation at 234 nm, after high-pressure liquid chromatography (HPLC) separation of phospholipid hydroperoxides. The radical initiators, water-soluble 2,2'-azobis(2-amidinopropane) (AAPH) and lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN), when thermally decomposed at 37 degrees C under air, produced a constant rate of lipid peroxidation in microsomes and lag times inversely related to their concentrations. Using 25 mM AAPH, beta-carotene suppressed lipid peroxidation at a concentration of 50 nmol/mg protein; using 24 mM AMVN, an inhibition of MDA formation was observed at a concentration of only 5 nmol/mg protein. Inhibition by beta-carotene did not produce a clearly defined lag phase. During AAPH-induced lipid peroxidation, beta-carotene was consumed linearly, and high levels of the antioxidant were still present at the end of 45 min of incubation. Using NADPH/ADP/Fe+3, protection by beta-carotene was observed at 10 nmol/mg protein. alpha-Tocopherol effectively suppressed both MDA and hydroperoxide formation in a dose-dependent manner when either NADPH/ADP/Fe+3 or azocompounds were used. These effects were observed at very low concentrations of the added alpha-tocopherol, ranging from 2 to 3 nmol/mg protein. When the lag times were measurable (AAPH and AMVN), they were directly proportional to the concentration of alpha-tocopherol and revealed the presence of endogenous antioxidants in the microsomal membranes. Different temporal relationships between the loss of alpha-tocopherol and lipid peroxidation were observed in relation to the prooxidant used. A substantial depletion of about 70% of endogenous alpha-tocopherol preceded the propagation phase when induced by the azocompounds, while only 20% of antioxidant disappeared at the beginning of the peroxidation when induced by NADPH/ADP/Fe+3. Although our results show that both beta-carotene and alpha-tocopherol suppress the peroxidation of microsomal membranes, their antioxidant efficacy is influenced by several factors, including the type of radical initiator involved and the site and rate of radical production.     

Activation of lipid peroxidation in the adrenal cortex by metal ions

The processes of lipid peroxidation have been studied in bovine adrenal cortex in vitro. The lipid peroxidation rate in this tissue is shown to be dependent on the content of metal ions. EDTA, deferroxamine and penicyllamine inhibit spontaneous lipid peroxidation by 25, 50 and 42%, respectively. The ability to activate the process permits arranging metal ions in the following sequence: Fe2+ greater than Fe3+ greater than Cu2+ greater than Mg2+ greater than Mn2+. The maximum activation of lipid peroxidation is observed at Fe2+ and Fe3+ concentrations within the range of 5 x 10(-6) x 10(-4) M.     

Antioxidant activity of synthetic diarylamines: a mitochondrial and cellular approach

We investigated the antioxidant properties of two synthetic diarylamines, MJQ1 and MJQ2. For one of them (MJQ1) the synthesis procedure is herein described. The compounds showed maximal protection of ADP/Fe(2+) induced mitochondrial lipid peroxidation for 50nM (MJQ1) and 60muM (MJQ2) concentrations. Both compounds were also effective in the prevention of mitochondrial DeltaPsi collapse. The effective antioxidant dose of MJQ1 in mitochondria (50nM) also proved to protect lipid peroxidation in PC12 cells and the effect seems not to be related with the compound's iron chelating ability. The modified structure of MJQ1 clearly resulted in an improvement of its antioxidant and toxic profile, evaluated in mitochondria and whole cells. This study demonstrates a high potential of these diarylamines, as radical scavengers, whose chemical structures can be manipulated if a specific target is well characterized.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.     

Plasma thiobarbituric acid reactivity: reaction conditions and the role of iron, antioxidants and lipid peroxy radicals on the quantitation of plasma lipid peroxides

The effects of Fe3+, lipid peroxy radicals and the antioxidant butylated hydroxytoluene on the 2-thiobarbituric (TBA) acid quantitation of plasma lipid peroxides were investigated. Whole plasma and plasma fractions prepared by trichloroacetic acid (TCA) protein precipitation and lipid extraction, demonstrated markedly differing TBA reactivities in the presence or absence of added Fe3+. Examination of the spectral profiles of the TBA reacted whole plasma and TCA precipitated fractions demonstrated the presence of interfering compounds which gave rise to an artifactual increase in lipid peroxide concentrations. In contrast the TBA reacted lipid extracts had low levels of interfering compounds that could be removed by our previously described high pressure liquid chromatographic method (Wade, Jackson and van Rij (1985) Biochem. Med. 33, 291-296). Further characterization of the TBA reactivity of the lipid extract showed that Fe3+ at an optimal concentration of 0.5 mM was necessary for the quantitative decomposition of the lipid peroxides to the TBA reactive product malondialdehyde (MDA). However the presence of Fe3+ resulted in further peroxidation of any unsaturated lipids present. Butylated hydroxytoluene (BHT) at an optimal concentration of 1.4 mM inhibited Fe3+ stimulated peroxidation without affecting the formation of the MDA-TBA chromogen. Using a standardized TBA test with plasma lipid extracts and the addition of optimal concentrations of Fe3+ and BHT, we have determined the mean concentration of lipid peroxides in 30 healthy human subjects to be 102.7 +/- 20.0 ngm/ml.