Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

Inhibition of lipoxygenase-dependent lipid peroxidation by quercetin: Mechanism of antioxidative function

Quercetin inhibited soybean lipoxygenase-1-dependent linoleic acid peroxidation. Two to three μM quercetin was required for 50% inhibition. During the inhibition, quercetin was oxidized. The oxidation was observed as an absorbance decrease at about 380 nm and an absorbance increase at about 335 nm. Inhibition of linoleic acid peroxidation by quercetin seems to be due to reduction by the reagent of the linoleic acid radical formed as an intermediate during lipoxygenation. Quercetin oxidation was suppressed by ascorbate under conditions when ascorbate did not affect lipoxygenase-dependent linoleic acid peroxidation. The results suggest that ascorbate can reduce the quercetin oxidized by the linoleic acid radical back to quercetin. Based on the results, the significance of a redox reaction between oxidized quercetin and ascorbate is discussed.     

Suppression of Carotenoid Photobleaching by Kaempferol in Isolated Chloroplasts

The effects of kaempferol on carotenoid photobleaching wereexamined using chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone(CCCP). Kaempferol suppressed carotenoid photobleaching withoutaffecting electron transfer reactions. Half-maximal suppressionwas observed at about 10 µM. Kaempferol was photooxidizedby CCCP-poisoned chloroplasts, as observed by its bleachingat 380 nm. Ascorbate inhibited the oxidation of kaempferol.Under anaerobic conditions, kaempferol did not affect the photobleachingof carotenoid. Other fiavonols, quercetin and its glycosides,also suppressed the carotenoid photobleaching. The results suggestthat flavonols act as antioxidants in illuminated chloroplastsunder aerobic conditions. (Received February 22, 1982; Accepted May 14, 1982)     

Redox Reactions between Kaempferol and Illuminated Chloroplasts

Bleaching of kaempferol by illuminated chloroplasts was observed at 380 nanometers. The photobleaching was stimulated by methyl viologen and suppressed by superoxide dismutase indicating the participation of O₂⁻ in the reaction. An electron transfer inhibitor on the oxidizing side of photosystem II, carbonylcyanide m-chlorophenylhydrazone (CCCP), stimulated the photobleaching and 3-(3,4-dichlorophenyl)-1,1-dimethylurea partially suppressed it. The stimulation by CCCP suggests that kaempferol is also bleached on the oxidizing side of photosystem II. The spectrum of kaempferol bleaching in the presence of methyl viologen was the same as that in the presence of CCCP having a maximum in absorbance decrease at around 380 nanometers. When kaempferol was oxidized by KMnO₂ or KO₂, the oxidized minus reduced difference spectra had also a negative peak at about 380 nanometers. The results suggest that kaempferol was oxidized by illuminated chloroplasts.

The rate of kaempferol photooxidation increased as its concentration was increased from 1 to 100 micromolar. The rate of quercetin photooxidation also increased as its concentration was increased from 1 to 100 micromolar. Concentration of quercetin glycosides higher than 10 micromolar was required to detect their photobleaching by illuminated chloroplasts. From these results, it is postulated that flavonols function as antioxidants in chloroplasts.

     

An essential difference between the flavonoids monoHER and quercetin in their interplay with the endogenous antioxidant network

Antioxidants can scavenge highly reactive radicals. As a result the antioxidants are converted into oxidation products that might cause damage to vital cellular components. To prevent this damage, the human body possesses an intricate network of antioxidants that pass over the reactivity from one antioxidant to another in a controlled way. The aim of the present study was to investigate how the semi-synthetic flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER), a potential protective agent against doxorubicin-induced cardiotoxicity, fits into this antioxidant network. This position was compared with that of the well-known flavonoid quercetin. The present study shows that the oxidation products of both monoHER and quercetin are reactive towards thiol groups of both GSH and proteins. However, in human blood plasma, oxidized quercetin easily reacts with protein thiols, whereas oxidized monoHER does not react with plasma protein thiols. Our results indicate that this can be explained by the presence of ascorbate in plasma; ascorbate is able to reduce oxidized monoHER to the parent compound monoHER before oxidized monoHER can react with thiols. This is a major difference with oxidized quercetin that preferentially reacts with thiols rather than ascorbate. The difference in selectivity between monoHER and quercetin originates from an intrinsic difference in the chemical nature of their oxidation products, which was corroborated by molecular quantum chemical calculations. These findings point towards an essential difference between structurally closely related flavonoids in their interplay with the endogenous antioxidant network. The advantage of monoHER is that it can safely channel the reactivity of radicals into the antioxidant network where the reactivity is completely neutralized.     

Oxidation products of quercetin catalyzed by mushroom tyrosinase

Quercetin was oxidized as a substrate catalyzed by mushroom tyrosinase to the corresponding o-quinone and subsequent isomerization to p-quinone methide type intermediate; followed by the addition of water on C-2 yielding a relatively stable intermediate, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. In the presence of a catalytic amount of l-DOPA as a cofactor, the rate of this oxidation was enhanced. Fisetin, which lacks the C-5 hydroxyl group, was also oxidized but the rate of oxidation was faster than that of quercetin, indicating that the C-5 hydroxyl group is not essential but is associated with the activity.     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

The Influence of Apoplastic Ascorbate on the Activities of Cell Wall-Associated Peroxidase and NADH Oxidase in Needles of Norway Spruce (Picea abies L.)

Cell wall-associated peroxidases (EC 1.11.1.7 [EC] ) were extractedfrom the current year's needles of Norway spruce trees (Piceaabies L.) in two fractions, namely soluble apoplastic peroxidasesand covalently wall-bound peroxidases. Peroxidase activitieswere determined with two substrates: coniferyl alcohol, whichis important for lignification, and NADH, which is necessaryfor the production of H₂O₂. Coniferyl alcohol peroxidase activitywas detected in both the soluble apoplastic fraction and thewall-bound fraction, whereas NADH oxidase activity was foundonly in the soluble apoplastic fraction. Net oxidation of coniferylalcohol and NADH was inhibited by ascorbate, which reduced theoxidized intermediates of the peroxidase- and oxidase-catalyzedreactions. Since ascorbate itself was oxidized in these reactions,the inhibition was not persistent and it was released once theascorbate present in the assay mixture had been oxidized. Ascorbatedelayed the oxidation of NADH 10-fold more efficiently thanthe oxidation of coniferyl alcohol. Although the level and theredox state of apoplastic ascorbate were lower in lignifyingneedles than in mature needles, the concentration, which was1.17 mM in apoplastic washing fluids, was sufficiently highto inhibit peroxidase activity in vitro. These results suggestthat peroxidases can catalyze lignification only if local differencesexist in the concentration of reduced ascorbate between lignifyingand non-lignifying tissues. (Received April 21, 1994; Accepted September 26, 1994)     

Inhibition Mode of Soybean Lipoxygenase‐1 by Quercetin

Quercetin noncompetitively inhibited the peroxidation of linoleic acid catalyzed by soybean lipoxygenase‐1 (EC 1.13.11.12, Type 1) with an IC₅₀ value of 4.8 μM (1.45 μg/ml). This inhibition is considered to proceed in sequential order, by initially reducing the ferric form of the enzyme to an inactive ferrous form and then, by chelating the iron of the active site of the enzyme. In the pseudoperoxidase assay, quercetin was slowly oxidized by hydroperoxides to a rather stable intermediate, 2‐(3,4‐dihydroxybenzoyl)‐2,4,6‐trihydroxybenzofuran‐3(2H)‐one, and this oxidized intermediate still inhibited the enzymatic oxidation, probably as a chelator. Rutin and kaempferol also exhibited lipoxygenase‐1 inhibitory activity, but to a much lesser extent than quercetin.     

Antioxidant Activity of Quercetin against Metmyoglobin-induced Oxidation of Fish Oil-bile Salt Emulsion

The antioxidative effect of quercetin was examined in metmyoglobin-induced oxidation of a fish oil-bile salt emulsion (average diameter of particles; 2.0 μm) to evaluate its effectiveness during the digestion of highly oxidizabile oils. The activity of quercetin increased with the lowering of the initial peroxide value (PV) of the oil and its effectiveness was superior to that of α-tocopherol. A synergistic antioxidant effect was observed upon the addition of quercetin and α-tocopherol irrespective of the initial PV of the oils, and quercetin was consumed faster than α-tocopherol. The loss of quercetin was larger than that of α-tocopherol when cumene hydroperoxide and metmyoglobin were mixed in a trimyristin-bile salt emulsion. In an ultrafiltration experiment on emulsified oil with a membrane filter of 100 nm pore size, the recovery of quercetin in the filtrate was higher than that of α-tocopherol. These data suggest that quercetin was an antioxidant in the digestion of fish oil. The effectiveness seems to come from its distribution in the emulsified oil, different from that of α-tocopherol, and its ability to scavenge radicals generated from the reaction of lipid hydroperoxides with metmyoglobin.     

Properties of Quercetin Conjugates: Modulation of LDL Oxidation and Binding to Human Serum Albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3′-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2?μM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3′-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide>quercetin>quercetin-3-glucuronide=quercetin-3-glucoside>catechin>quercetin-4′-glucuronide>isorhamnetin-3-glucuronide>quercetin-3′-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3′-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3′-sulfate>quercetin-7-glucuronide>quercetin-3-glucuronide>quercetin-4′-glucuronide=isorahmnetin-3-glucuronide). K_q values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3′-sulfate (～4?μM)=quercetin≥quercetin-7-glucuronide>quercetin-3-glucuronide=quercetin-3-glucoside>isorhamnetin-3-glucuronide>quercetin-4′-glucuronide (～20?μM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox active copper binding: relevance to diseases of glycoxidative stress.

Aging and age-related diseases are associated with the production of reactive oxygen species which modify lipids, proteins and DNA. Here we hypothesized the glyco- and lipoxidation product N(epsilon)-(carboxymethyl)lysine (CML) in proteins should bind divalent and redox active transition metal binding. CML-rich poly-L-lysine and bovine serum albumin (BSA) were chemically prepared and found to bind non-dialyzable Cu(2+), Zn(2+) and Ca(2+). CML-BSA-copper complexes oxidized ascorbate and depolymerized protein in the presence of H(2)O(2). CML-rich tail tendons implanted for 25 days into the peritoneal cavity of diabetic rats had a 150% increase in copper content and oxidized ascorbate three times faster than controls. CML-rich proteins immunoprecipitated from serum of uremic patients oxidized four times more ascorbate than control and generated spin adducts of DMPO in the presence of H(2)O(2). The chelator DTPA suppressed ascorbate oxidation thereby implicating transition metals in the process. In aging and disease, CML accumulation may result in a deleterious vicious cycle since CML formation itself is catalyzed by lipoxidation and glycoxidation.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

Inhibition of Mammalian 15-Lipoxygenase-Dependent Lipid Peroxidation in Low-Density Lipoprotein by Quercetin and Quercetin Monoglucosides

Lipoxygenase is suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation in the subendothelial space of the arterial wall. Since flavonoids such as quercetin are recognized as lipoxygenase inhibitors and they occur mainly in the glycoside form, we assessed the effect of quercetin and its glycosides (quercetin 3-O-β-glucopyranoside, Q3G; quercetin 4′-O-β-glucopyranoside, Q4′G; quercetin 7-O-β-glucopyranoside, Q7G) on rabbit reticulocyte 15-lipoxygenase (15-Lox)-induced human LDL lipid peroxidation and compared it with the inhibition obtained by ascorbic acid and α-tocopherol, the main water-soluble and lipid-soluble antioxidants in blood plasma, respectively. Quercetin inhibited the formation of cholesteryl ester hydroperoxides (CE-OOH) and endogenous α-tocopherol consumption effectively throughout the incubation period of 6 h. Ascorbic acid exhibited an effective inhibition only in the initial stage and LDL preloaded with fivefold α-tocopherol did not affect the formation of CE-OOH compared with the native LDL. CE-OOH formation was inhibited by both quercetin and quercetin monoglucosides in a concentration-dependent manner. Quercetin, Q3G, and Q7G exhibited a higher inhibitory effect than Q4′G (IC₅₀: 0.3–0.5 μM for quercetin, Q3G, and Q7G and 1.2 μM for Q4′G). While endogenous α-tocopherol was completely depleted after 2 h of LDL oxidation, quercetin, Q7G, and Q3G prevented the consumption of α-tocopherol. Quercetin and its monoglucosides were also exhausted during the LDL oxidation. These results indicate that quercetin glycosides as well as its aglycone are capable of inhibiting lipoxygenase-induced LDL oxidation more efficiently than ascorbic acid and α-tocopherol.     

Oxidation of Flavonols by Hydrogen Peroxide in Epidermal and Guard Cells of Vicia faba L.

Epidermal strips of Vicia faba were found to contain kaempferoland quercetin glycosides. These flavonols were oxidized by H₂O₂and oxidation was inhibited by KCN (3.5 nM). Quercetin glycosideswere more sensitive to H₂O₂ than kaempferol glycosides. Oxidationcould be detected in epidermal strips even at 30 µM H₂O₂.Flavonol oxidation by H₂O₂ was observed in both guard and epidermalcells. In guard cells, oxidation appeared as the bleaching ofabsorption bands of flavonols. Epidermal cells could be roughlydivided into two types on the basis of their absorption characteristicsin the UV-light region. In one type, only flavonol oxidationwas observed; in the other, both flavonol and 3,4-dihydroxyphenylalanine(DOPA) oxidation were observed. Oxidation of flavonols and DOPAby H₂O₂ was also observed in cell-free extracts of the epidermalstrips, even at 10µ H₂O₂. Oxidation was inhibited by 1mM KCN, suggesting the participation of peroxidase in the reactions.The data obtained in this study indicate the cellular specificdistribution of phenolic compounds in the epidermis and thepossibility of their oxidation by H₂O₂ generated in epidermaland guard cells. (Received August 24, 1987; Accepted January 21, 1988)     

Hydrogen Peroxide-Dependent Oxidation of Flavonoids and Hydroxycinnamic Acid Derivatives in Epidermal and Guard Cells of Tradescantia virginiana L.

Luteolin, kaempferol, quercetin, caffeic acid and ferulic acidwere identified in acid-hydrolyzed epidermal strips of Tradescantiavirginiana using HPLC and spectrophotometry. The amount of flavonoidswas much smaller than that of cinnamic acid derivatives. Morethan 80% of the flavonoids were found in methanol extracts ofepidermal strips. Caffeic acid was found in both methanol extractsand the residues in nearly equal amounts, while more than 80%of the ferulic acid was found in the residues after methanolextraction. These data suggest that most of the ferulic acidand part of the caffeic acid bind to macromolecules as estersin the cell wall and that flavonoids are localized mainly inthe cytoplasm. The localization of esters of hydroxycinnamicacids in cell walls was ascertained by fluorometric analysis.These phenolic compounds were oxidized by H₂O₂ (0.025–1mM) in epidermal and guard cells and the oxidation was inhibitedby KCN and NaN₃: luteolin glycosides were less sensitive toH₂O₂ than quercetin and kaempferol glycosides in flavonoids.Ferulic acid esters were more sensitive to H₂O₂ than caffeicacid esters in hydroxycinnamic acid derivatives. On the basisof these data, the physiological significance of the oxidationof phenolic compounds by H₂O₂ is discussed. (Received October 9, 1987; Accepted February 3, 1988)     

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.     

A Possible Mechanism for the Oxidation of Sinapyl Alcohol by Peroxidase-Dependent Reactions in the Apoplast: Enhancement of the Oxidation by Hydroxycinnamic Acids and Components of the Apoplast

Apoplastic peroxidase isoenzymes from stems of Nicotiana tabacumrapidly oxidized sinapic acid and sinapyl alcohol, in additionto 4-coumaric acid, ferulic acid and coniferyl alcohol. By contrast,the peroxidase isoenzymes from stems of Vigna angularis oxidizedsinapic acid and sinapyl alcohol quite slowly but rapidly oxidizedcompounds with a 4-hydroxyphenyl or a guaiacyl group. However,the oxidation of sinapyl alcohol was greatly enhanced by 4-coumaricacid, ferulic acid and an ester of ferulic acid. Intercellularwashing fluid of V. angularis, which contained apoplastic components,also enhanced the oxidation of sinapyl alcohol. Based on theseresults, a possible mechanism for the oxidation of sinapyl alcoholis discussed on the assumption that the biosynthesis of ligninproceeds mainly via peroxidases which cannot oxidize sinapylalcohol in V. angularis. (Received October 23, 1995; Accepted April 3, 1996)     

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.