219

Redox Cycling of Iron and Lipid Peroxidation 1 Giorgio Minotti a and Steven D. Aust b,* alnstitute of General Pathology, Catholic University School of Medicine, 00168 Rome, Italy and bBiotechnology Center, Utah State University, Logan, Utah 84322-4700

M e c h a n i s m s 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 prom o t e the formation of new LOOH. However, in the absence of LOOH, both Fe 2+ and Fe 3+ m u s t be available to initiate lipid peroxidation, with o p t i m u m activity occurring as the Fe2+/Fe 3+ ratio approaches unity. The s i m u l t a n e o u s availability of Fe 2+ and Fe 3+ can be achieved by oxidizing some Fe 2+ with hydrogen peroxide or with chelators that favor autoxidation of Fe 2§ by molecular oxygen. Alternatively, one can use Fe 3§ and reductants like superoxide, ascorbate or thiols. In either case excess Fe z+ oxidation or Fe 3+ reduction will inhibit lipid peroxidation by converting all the iron to the Fe 3§ or Fe 2§ form, respectively. Superoxide dismutase and catalase can affect lipid peroxidation by affecting iron reduction/oxidation and the formation of a (1:1) Fe2+/Fe 3+ ratio. H y d r o x y l radical scavengers can also increase or decrease lipid peroxidation by affecting the redox cycling of iron. Lipids 27, 219-226 (1992).

Lipid peroxidation is the process in which molecular oxygen is incorporated into unsaturated lipids (LH) to form lipid hydroperoxides (LOOH). However, the direct reaction of lipids with oxygen is spin-forbidden because the ground state of lipids is of singlet multiplicity whereas t h a t of oxygen is of triplet multiplicity (1). Lipid peroxidation m u s t therefore occur via reactions t h a t by-pass the spin barrier between lipids and oxygen. These reactions are promoted by some type of "initiator" (I ~ t h a t overcomes the dissociation energy of an allylic bond and t h u s causes hydrogen abstraction and formation of a lipid alkyl radical (L'). Lipid alkyl radicals can rapidly add oxygen to form lipid peroxyl radicals (LOO') which eventually liberate L O O H via hydrogen abstraction from a neighboring allylic bond (2). I" + L H - " I H + L"

initiation

[1]

L" + O2 -* LOO"

02 addition

[2]

LO0" + L H -* L O O H + L"

propagation

[3]

IBased on a paper presented at the Symposium on Metals and Lipid Oxidation, held at the AOCS Annual Meeting in Baltimore, Maryland, April 1990. *To whom correspondence should be addressed. Abbreviations: ADP, adenosine diphosphate; EDTA, ethylenediaminetetraacetic acid; "OH, hydroxyl radical; L ~ lipid alkyl radical; LH, unsaturated lipids; LOOH, lipid hydroperoxides; LOO', lipid peroxyl radicals; MDA, malondialdehyde; NADPH, reduced nicotinamide adenine dinucleotide phosphate; O2", superoxide; SOD, superoxide dismutase.

Externally generated oxidants are required to initiate lipid peroxidation, b u t once started, the reaction will proceed via propagation to form new LOOH. Transition metals can substantially enhance the propagation of lipid peroxidation. For example, Fe 2+ will reductively cleave LOOH to highly reactive alkoxyl (LO') radicals, which in turn abstract hydrogen from lipids to form new lipid alkyl radicals (3). This reaction is referred to as LOOHdependent lipid peroxidation (3). Fe 2+ + L 0 0 H ~ Fe a+ + L0" + O H -

[4]

LO" + L H --" L O H + L"

[5]

The realization t h a t numerous diseases may involve lipid peroxidation as a common pathogenic mechanism (4) has prompted interest in identification of possible initiators (see Eq. 1). Both iron and partially reduced species of dioxygen have been implicated in the formation of initiators, although with very different interpretations concerning the mechanism of their involvement. One very popular theory dictates t h a t lipid peroxidation is initiated by "OH (5-8), the most potent oxidant t h a t can be formed from oxygen (E ~ = 1.6 V) (9). This is thought to occur via the Haber-Weiss reaction, that consists of 02" dependent Fe 3+ reduction and subsequent Fe2+-catalyzed H202 cleavage to "OH (10). 02" + Fe 3+ ~ 02 + Fe 2+

[6]

20~" + 2H + --* 2H202 + 02

[71

Fe 2+ + H202 --* Fe 3+ + O H - + "OH

[81

Ferrous iron-promoted breakdown of H202 (Eq. 8) is best known as the Fenton reaction, and the Haber~Weiss reaction may alternatively be referred to as 02" driven Fenton reaction, with this terminology illustrating t h a t O2" is required for the formation of both Fe 2+ (via reduction of Fe 3+, Eq. 6) and H202 (via dismutation, Eq. 8) (10). According to this interpretation, the inhibition of lipid peroxidation by superoxide dismutase (SOD), catalase or hydroxyl radical ('OH) scavengers is viewed as supporting evidence of initiation of lipid peroxidation via Fenton chemistry and the "OH (6,7) As an alternate proposal for the initiation of lipid peroxidation however, some investigators have proposed that iron oxygen complexes can substitute for "OH in the initiation of lipid peroxidation, provided t h a t both Fe 2+ and Fe 3+ are involved in the formation of this complex (11-20). This alternate interpretation implies t h a t O2" and H202 are required not to form ~ but rather to promote the Fe ~+ reduction or Fe 2+ oxidation from which the appropriate Fe2+/Fe3+ initiating species originates. Consistent with this proposal, the effects of SOD, catalase and "OH scavengers have been found to v a r y depending on their effects on the iron valence s t a t e (15,20). F u r t h e r m o r e , 0 2 " and H202 LIPIDS, Vol. 27, no. 3 (1992)

220

REVIEW independent mechanisms for the redox cycling of iron and the initiation of lipid peroxidation have been described (18,20). In this article we describe and discuss some mechanisms for the redox cycling of iron and their relevance to the initiation of lipid peroxidation by either "OH or equally effective iron/oxygen complexes. FERROUS IRON OXIDATION AND LIPID PEROXIDATION

Lipid peroxidation can be studied in incubations containing unchelated Fe 2+ and either commercially available fatty acids or microsomal phospholipid liposomes. Ferrous iron p e r se may promote or not promote lipid peroxidation, depending on whether lipids contain preformed LOOH. In the presence of preformed LOOH, Fe2+ will catalyze what we have previously referred to as LOOHdependent lipid peroxidation (3), a reaction resembling propagation rather than initiation mechanisms. In the absence of preformed LOOH, or when the concentration of LOOH is so low that they may not react with Fe 2+ to an appreciable rate, H202 can be used to promote Fenton reaction and "OH formation. We observed that in model systems including FeC12 and microsomal phospholipid liposomes with very little, if any, LOOH contamination, the inclusion of H202 promoted lipid peroxidation, as evidenced by formation of malondialdehyde {MDA) {Table 1). The reaction was inhibited by catalase, but the addition of "OH scavengers either inhibited or stimulated MDA formation. These effects were not due to the ability of mannitol and benzoate to scavenge "OH, but could be explained by the ability of these compounds to interfere with Fe 2+ oxidation. Thus, lipid peroxidation was inhibited by mannitol, which minimizes Fe3+ formation,

whereas lipid peroxidation was stimulated by benzoate, which facilitates Fe 3+ formation (see also Table 1). Collectively, these observations have led us to question the involvement of "OH in lipid peroxidation and the effects of "OH scavengers therein. We have proposed that: (i) Fe 2+ dependent peroxidation of LOOH-free lipids is mediated by an oxidant that also requires Fe3+, (ii) the role of H 2 O 2 is that of oxidizing Fe2+ to Fe3+ rather than of serving as precursor of "OH, and (iii) chemicals referred to as "OH scavengers must be used with great caution, since they can interfere with lipid peroxidation by modulating the rate of Fe 2+ oxidation. In principle, our skepticism concerning the participation of "OH in lipid peroxidation agrees with the criticism that "OH is a short-lived radical with diffusion-limited reactivity (21). As such, "OH would not migrate from the aqueous phase of incubations to the hydrophobic membrane compartments where the allylic bonds of fatty acids are buried. For example, any "OH generated in the aqueous bulk of incubations can react with residual Fe2+ and H202 (Eqs. 9-10) or can undergo bimolecular recombination to water (Eq. 11), thus self-precluding from reaction with lipids (8,9). "OH + Fe2+ + H + --" Fe~+ + n 2 0

[9]

"OH + H202" "* HO 2" + H20

[10]

"OH + "OH -~ H 2 0 2

[11]

However, Schaich and Borg (8,22) have found that critical amounts of iron and H202 can partition from water into lipids. They have therefore proposed that Fenton reaction may take place in the lipids, so that "site specific" reaction of "OH with juxtaposed allylic bonds would prevail over nonproductive side reactions in the aqueous phase. Viewed in this context, stimulation or inhibition of lipid TABLE 1 peroxidation by benzoate or mannitol might simply reflect spurious and diverging effects of these compounds on Effects of Catalase, "OH Scavengers and Detergents on Fe 2+ "OH-dependent reactions that occur in the aqueous phase and H202 Dependent Lipid Peroxidationa, b and either "consume" Fe 2+ and H=O= (cf Eqs. 9-10) or evolve H202 (cf Eq. 11). Mannitol and benzoate would afFe2+ oxidation System (%) nmol MDA/mL/min fect lipid peroxidation by affecting the concentration of Fe2+ and H20= that migrate from water to lipids and proFe 2+ 0 0.0 mote the intramembranous formation of "OH. Fe 2+, H202 50 3.8 In order to validate or disprove this hypothesis, we have + catalase 0 0.0 studied Fe 2+ and H202-dependent peroxidation of phos+ mannitol 30 0.7 pholipid liposomes dispersed in nonionic detergent Lubrol+ benzoate 85 5.5 PX. In this system, the detergent served to modify lipid Fe 2+, H202, Lubrol-PX 53 6.1 configuration and facilitated the partitioning of both iron + catalase 0 0.0 and "OH scavengers from water to lipids (6,23). This would + mannitol 39 2.8 facilitate the formation of "OH in closest proximity of the -- benzoate 92 8.2 allylic bond, thus minimizing reactions of "OH in the a Incubations contained microsomal phospholipid liposomes (1 ~ n o l aqueous phase and nonspecific interferences of "OH traps lipid phosphate/mL) _+ 0.1% Lubrol-PX, in 50 m M NaC[, p H 7.0, therein. As shown in Table 1, Lubrol-PX caused a substan37~ Where indicated, additions were made as follows: FeC12, 0.2 tial increase of lipid peroxidation, although it did not mM; H202, 0.1 raM; catalase, 400 U/mL; mannitol, 25 raM; benmodify the extent of Fe 2+ oxidation. This indicates that zoate, 25 mM. Ferrous iron oxidation was monitored as disappearance of 1,10-phenanthroline chelatable Fe 2+ in lipid-free inLubrol-PX stimulates lipid peroxidation mainly by incubations and values are those determined at 30 see. NaCl was creasing the accessibility of iron to lipids. However, the chromatographed on Chelex-100 to remove contaminating metals, effects of catalase" mannitol and benzoate on Fe 2+ oxidaand catalase was chromatographed on Sephadex G-25 to remove tion and formation of MDA were the same as observed the antioxidant thymol and other contaminants. M D A formation in incubations lacking the detergent (see also Table 1). was determined by thiobarbituric acid test within the linear phase of the reactions (4-6 min.). Therefore, we must reiterate the earlier contention that the initiation of lipid peroxidation by Fe2+ is mediated by b D a t a t a k e n in p a r t from Minotti and A u s t (15). LIPIDS, Vol. 27, no. 3 (1992)

221

REVIEW TABLE 2 Effect of Preincubating Fe 2+ with H202 on Fe 2+ Oxidation and Lipid Peroxidation a, b

Preincubation time (sec)

Fe 2+ oxidation (%)

nmol MDA/mL/min

0 15 30 60 120 240

0 39 50 68 88 100

0.0 3.9 4.3 2.7 0.9 0.0

aHydrogen peroxide (0.1 mM) and FeCl 2 (0.2 mM) were preincubated in 50 mM NaC1, pH 7.0, 37~ for the time required to achieve the indicated extents of Fe 2+ oxidation, measured as described in legend to Table 1. At the end of preincubation, catalase (400 U/mL) was included to scavenge any remaining H202, and microsomal phospholipid liposomes (1 pmol lipid phosphate/mL) were included to study lipid peroxidation. bData from Minotti and Aust (15).

oxidants t h a t provide Fe 3+. Thus, in this s y s t e m H202 m u s t be available to ensure Fe 2+ oxidation, whereas mannitol and benzoate will modulate lipid peroxidation by decreasing or increasing Fe 3+ formation, irrespective of their ability to intercept "OH in either aqueous or lipid environments. To quantitate how m u c h Fe 3+ is required for the initiation of lipid peroxidation using Fe 2+, we studied liposome incubations containing various a m o u n t s of Fe 2+ and Fe 3+ generated by reacting Fe 2+ with H202 for increasing periods of time. As shown in Table 2, the rate of lipid peroxidation was highest when approximately 50% of Fe 2+ had been oxidized to Fe 3+. Lipid peroxidation did not occur when all of the iron remained in the reduced form nor when H202 had oxidized all the Fe 2+ to Fe 3+. These results suggested to us t h a t only a fraction of Fe 2+ m u s t be converted to Fe 8+, with o p t i m u m activity occurring as the Fe2+/Fe 3+ ratio approaches unity. FERROUS IRON AUTOXIDATION AND LIPID PEROXIDATION The autoxidation of Fe 2+ m a y represent an alternate m e a n s to generate Fe 3+ and thus initiate lipid peroxidation. Iron autoxidation consists of direct electron transfer from Fe 2++ to molecular oxygen, leading to formation of 02" and Fe 3+ (Eq. 12). Alternatively, and perhaps more likely (1), two Fe 2+ can autoxidize at the expense of one molecule of oxygen, thus causing the formation of H202 and two Fe 3+ (Eq. 13). Fe 2+ + 02 --~ Fe 3+ + 0 2"

[12]

2Fe 2+ + 02 + 2H + --~ 2Fe 3+ + H202

[13]

In order for iron to autoxidize, the reduction potential of the Fe2+/Fe 3+ couple m u s t be lower t h a n t h a t of the 02"/02 couple (-0.33 V) (24). I t is generally a s s u m e d t h a t the redox potential of Fe2+/Fe 3+ couple is - 0 . 7 7 V (25) and t h a t chelators with oxygen donor a t o m s and greater affinity for Fe 3+ lower this value (26). However,

- O H ligation of the iron m a y p e r s e lower the redox potential of Fe 2+ by a b o u t 0.66 V, t h u s m a k i n g the autoxidation of "free" Fe 2+ theoretically feasible (1). However, even in such a case, chelators will dictate the rate of iron autoxidation. For example, unchelated Fe 2+ and ethylenediaminetetraacetic acid (EDTA) chelated Fe 2+ have a similar redox potential (-0.11 V) (1), yet FeSO4 or FeC12 will autoxidize very slowly whereas EDTA/Fe 2+ will autoxidize very rapidly, provided t h a t these reactions are studied at neutral p H and in an inert solution like NaC1 (27}. I t is a frequent misconception t h a t unchelated Fe 2+ autoxidizes rapidly, b u t this contention does not take into account t h a t several laboratory buffers chelate iron and modify its redox chemistry, u l t i m a t e l y favoring Fe 2+ autoxidation. P h o s p h a t e buffers are perhaps the m o s t effective in causing Fe 2+ autoxidation (1), and this m a y explain some conflicting reports in the literature. For example, we have already mentioned t h a t unchelated Fe 2+ cannot peroxidize LOOH-free liposomes incubated in NaC1, b u t others have shown t h a t Fe 2+ m a y quite effectively p r o m o t e the peroxidation of liposomes incubated in p h o s p h a t e buffers (28). Irrespective of different L O O H contaminations of liposomes prepared in different laboratories, it is quite possible t h a t lipid peroxidation in phosphate-buffered incubations is mediated by Fe 2+ autoxidation and formation of the Fe 3+ also required for initiation rather t h a n by decomposition of L O O H by Fe 2+ a l o n e There are some basic aspects of ligand-affected iron autoxidation t h a t m u s t be kept in mind for rigorous interpretation of lipid peroxidation experiments. First of all, iron autoxidation increases with the chelator/Fe ~+ r a t i ~ with the extent of autoxidation strictly depending on the chelator being used. For example, EDTA/Fe 2+ autoxidizes much more rapidly t h a n citrate/Fe 2+ and the autoxidation of approximately 50-70% of the iron will therefore occur at v e r y different chelator/Fe 2+ ratios (1:1 for EDTA/Fe 2+ v s 20:1 for citrate/Fe 2+) (16,27). Second, in the presence of ligands Fe 2+ autoxidation m a y not be significantly inhibited by catalase (16,20,27). Direct consequences of these m e c h a n i s m s of iron autoxidation on the initiation of lipid peroxidation are exemplified in Table 3. The d a t a indicate t h a t b o t h citrate/Fe 2+ and TABLE 3 The Effects of Chelators on Iron oxidation and Lipid Peroxidation a, b

Chelate

Fe2+ oxidation (%)

nmol MDA/mL/min

- catalase + catalase - catalase + catalase EDTA/Fe2+ (1:1) 75 63 1.4 1.3 (20:1) 100 100 0.0 0.0 Citrate/Fe 2+ (1:1) 19 16 0.3 0.4 (20:1) 63 48 1.0 1.2 a Incubations contained microsomal phospholipid liposomes (1/#tool lipid phosphate/mL) in 50 mM NaC1, pH 7.0, 37~ Reactions were started by addition of chelates at different chelator/FeC12 ratios. The concentration of Fe2+ was 200 ~M. Where indicated, catalase (400 U/mL) was included. Iron oxidation was measured as described in the legend to Table 1 and values are those determined at 30 sec. MDA formation is expressed as initial rates. bData from Minotti and Aust (16,27). LIPIDS, VoI. 27, no. 3 (1992)

222

REVIEW E D T A / F e e+ c a t a l y z e d lipid p e r o x i d a t i o n , y e t the EDTA/Fe e+ ratio had to be 1:1 and the citrate/Fe z+ ratio had to be 20:1. This is because lipid peroxidation occurred when only a critical fraction of Fe e+ was converted to Fe 3+. Very little, if any, lipid peroxidation occurred with either 20:1 EDTA/Fe e+ or 1:1 citrate/Fe z+ ratios, which caused too extensive or too moderate autoxidation, respectively, and thus shifted the Fee+/Fe 3+ ratios toward values t h a t were not permissive to lipid peroxidation (see also Table 3). By contrast, EDTA and citrate/Fe e+ dependent lipid peroxidation were largely insensitive to the addition of catalase, as were the autoxidation rates of the two chelates (Table 3). This underscored a substantial difference from systems in which H202 dependent and hence catalase-inhibitable oxidation of unchelated Fe e+ was m a n d a t o r y to form the necessary Fe 3+ (cf Table 1). The observation t h a t catalase cannot affect iron autoxidation must be examined from different viewpoints. One possibility might be that the vast majority of Fe 3+ forms via Eqs. 12-13, with very minor contribution by subsequent reactions of residual Fe e+ and the HeO 2 formed by oxygen reduction. Another possibility might be t h a t H202 does participate in Fe 3+ formation yet the access of catalase to the site of reaction between Fe e+ and HeO e is made sterically unfavorable by some type of "cage effect" (29) of the chelator being used. A l t h o u g h speculative in nature, this latter possibility should not be neglected, because we have found t h a t exogenously added H202 can react with citrate/Fe e+ and t h a t simultaneous addition of catalase prevents this reaction (16). Therefore, one may not rule out t h a t ligandaffected Fe e+ autoxidation would be paralleled by H202 dependent Fe e+ oxidation (see Table 3). Irrespective of any further speculation on the difference between exogenously added or endogenously formed HeO 2 for reaction with chelated iron, it is noteworthy t h a t the addition of H202 can either stimulate or inhibit citrate/Fe z+ dependent lipid peroxidation, depending on the citrate/Fe z+ ratio. With a low citrate/Fe ~+ ratio and very moderate Fe 2+ autoxidation, the addition of HeO2 was found to expedite Fe 3+ formation and approach Fee+/Fe3+ ratios that favored lipid peroxidation (see Table 4). With a high citrate/Fe z+ ratio and more extensive Fe e+ autoxidation,

TABLE 4 Effects of H202 on Citrate/Fe 2+ Dependent Lipid Peroxidation a, b Citrate/Fe e+ ratio 1:1

20:1

System

nmol MDA/mL/min

Complete + HeO2 +H202 + catalase Complete + HeO2 +H20 + catalase

0.3 1.3 0.4 1.4 0.3 1.3

% Change + 333 +33 -79 -7

a Complete systems contained citrate (at the indicated citrate/Fe 2+ ratios} and microsomal phospholipid liposomes [1 ~mol lipid phosphate/mL) in 50 mL NaC1, pH 7.0, 37~ Where indicated, 10 ~M H202 and catalase (400 U/mL) were included in the reaction mixtures. Fe2+ concentration was 200 ~M. Values of MDA formation are given as initial rates. bData from Minotti and Aust (16). LIPIDS, Vol. 27, no. 3 (1992)

the addition of H20 2 caused the formation of too much Fe 3+, thereby shifting the Fe2+/Fe 3+ ratio toward values t h a t were not permissive to lipid peroxidation (Table 4). In either case, the simultaneous addition of catalase prevented the reaction of H202 with citrate/Fe 2+, changed the Fe2+/Fe 3+ ratio, and either stimulated or inhibited lipid peroxidation. These results confirmed the role of H202 ill forming Fe 3+ from Fe 2+, with ultimate effects on lipid peroxidation depending on the ratio of Fee+/Fe 3+. Some investigators have noted t h a t unchelated Fe e+ can very rapidly promote lipid peroxidation of "intact" microsomes, irrespective of the presence or absence of an oxidant like HeO e or chelators favoring the autoxidation of Fe e+ [28). The occurrence of lipid peroxidation in incubations containing microsomes and Fe e+ but neither H20 e or chelators has been taken as evidence against the requirement for both Fe e+ and Fe 3+, which was first observed in liposomal systems. In this respect, Table 5 shows t h a t all of the exogenously added FeSO4 was recovered from incubations containing liposomes but not from incubations containing microsomes from which these same liposomes have been prepared. Microsome-induced ferrous iron disappearance was inhibited by anaerobiosis but not by catalase, as was the autoxidation of EDTA/ Fe e+ or citrate/Fe e+ (cfi Table 3 and Table 5). It is difficult to propose t h a t Fe e+ would react with preformed L O O H of "intact" microsomes but not of their extracted lipids. We would rather propose t h a t microsomes, but not liposomes, favor reaction of Fe e+ with oxygen and formation of the Fe 3+ required for initiation of lipid peroxidation (see Table 5). Studies are in progress to identify the nonlipid microsome components t h a t cause iron oxidation. Irrespective of the precise nature of these components, however, it is imperative to recognize microsome-induced Fe e+ oxidation as an additional mechanism for Fe 3+

TABLE 5 Iron Oxidation and Lipid Peroxidation: Comparison of Microsomal v s Liposomal Systems a

System Fe2+ + liposomes Fe2+ + microsomes Fe2+, microsomes, catalase Fe 2+ + microsomes (anaerobic)

Fe2+ oxidation (%)

nmoles MDA/mL/min

4 48

0.0 1.2

46

1.2

3

0.1

a Incubations contained FeSO4 (60 ~M) and either microsomes (0.6 mg proteirdmL) or microsomalphospholipid liposomes (0.6 ~nol lipid phosphate/mL) in 50 mM NaC1, pH 7.0, 37 ~ In experiments with microsomes, iron oxidation was measured at 5 min as disappearance of bathophenanthroline chelatable Fe2+ in 105,000 X g supernatants of incubations, as described (20}. In experiments with liposomes, aliquots of incubations were mixed with bathophenanthroline and formation of bathophenanthroline/Fe 2+ complex was determined spectrophotometrically upon addition of Tween 80 to eliminate turbidity, as described in (17). Where indicated, incubations were made anaerobic by means of argon plus glucose (5 mM) and glucose oxidase (10 U/mL); catalase was also included to scavenge H202produced by glucose oxidase reaction. Microsomes were calcium-aggregated and chromatographed on Sepharose CL-2B to remove contaminants like SOD, catalase and ferritin (see also ref. 20). Values of MDA formation are given as initial rates.

223 REVIEW formation and lipid peroxidation. This will aid the understanding and interpretation of results obtained b y different investigators under various experimental conditions. FERRIC IRON REDUCTION AND LIPID PEROXIDATION

Lipid peroxidation can also be studied in incubations containing ferric iron and reductants. One very popular system of lipid peroxidation relies on xanthine oxidase and adenosine diphosphate (ADP)/Fe 3+ (30). The xanthine oxidase reaction generates 02" as a reductant for Fe 3+. However, the xanthine oxidase reaction also generates H202, either via 0~" d i s m u t a t i o n or via the direct twoelectron reduction of molecular oxygen (31). Therefore, xanthine oxidase dependent generation of an iron reduct a n t is accompanied by the generation of an iron oxidant. Keeping in mind t h a t lipid peroxidation requires b o t h Fe 2+ and Fe 3+, one can easily anticipate t h a t xanthine oxidase and ADP/Fe 3+ dependent lipid peroxidation will be inhibited by SOD, which prevents Fe 8+ reduction, and will be enhanced by catalase, which prevents Fe 2+ oxidation back to Fe 8+ (Table 6). We have determined t h a t xanthine oxidase dependent generation of 02" at a rate of a p p r o x i m a t e l y 50 flM/min will reduce 100 gM ADP/Fe 3+ at a rate of 6 or 22 tAVI/min, depending on whether catalase is o m i t t e d or included to scavenge H202, respectively (32). This quantitation confirms t h a t the inclusion of xanthine oxidase with Fe 3+ rapidly m a k e s b o t h Fe3+ and Fe 2+ available to lipids, especially when catalase is included to prevent Fe 2+ reoxidation. The relatively high stoichiometry of 02" generation vs Fe 2+ formation (~5:1) t h a t persists even in the presence of catalase indicates t h a t some Fe 2+ invariably oxidizes to Fe 3+. I t is possible t h a t ADP/Fe 8+ reduction results in the f o r m a t i o n of an TABLE 6 Ferric Iron-Dependent Lipid Peroxidation: Comparison of Xanthine Oxidase v s Glutathione and Ascorbate Systems a

System Xanthine oxidaseb + SOD + catalase Glutathionec + SOD + catalase Ascorbated + SOD + cataiase

nmol MDA/mL/min 1.2 0.3 1.6 1.3 1.4 1.3 0.7 0.7 0.7

% Change --75 +33 +8

a Incubations contained microsomal phospholipid liposomes (1 tanol lipid phosphate/mL) in 50 mM NaC1, pH 7.0, 37~ Lipid peroxidation was assayed as malondialdehyde (MDA) formation (3). bSystem contained 0.11 mM ADP, 0.1 mM FeC12, 0.33 mM xanthine and 0.05 U/mL xanthine oxidase. SOD and catalase were added at 100 and 400 U/mL, respectively (27). Xanthine oxidase was chromatographed on Sephadex G-25 to remove ammonium sulfate and other contaminants. cSystem contained 0.11 mM ADP, and 0.1 mM FeC13 and 1 mM glutathione. SOD and catalase were added at 100 and 400 U/mL, respectively {27). dSystem contained 0.25 mM ADP, 0.05 mM FeC13 and 12.5 gM ascorbate. SOD and catalase were added at 15 U/mL (17).

ADP]Fe 2+ complex, which in t u r n autoxidizes in a catalase-insensitive manner. Nonetheless, it should also be noted t h a t ADP/Fe 2+ autoxidizes quite moderately as compared to other chelates, e.g., citrate/Fe 2+ or EDTA/ Fe 2+ (cf. refs. 16,18,27}. Therefore ADP/Fe 2+ autoxidation m a y not preclude formation of an appropriate Fe2+/Fe 8+ ratio. Another possibility is t h a t O~" m a y also serve as an oxidant for Fe 2+, thereby m a i n t a i n i n g iron within a continuous Fe 3+ -* Fe 2+ ~ Fe 8+ cycle {1,32}. T h o m a s and A u s t (33) and Saito e t al. (34) have described ferritin- and transferrin-dependent lipid peroxidation s y s t e m s in which xanthine oxidase is required to form O2" and promote the reductive release of iron from these proteins. As a general feature, rates of iron release are less t h a n one-tenth or one-fifth of rates of ADP/Fe 3+ reduction (cf refs. 32-34). Therefore. catalase m u s t be included to prevent H202 from causing the complete oxidation of the released Fe 2+ and inhibiting the formation of a suitable Fe2+/Fe 3+ ratio. The Fe 3+ required to combine with Fe 2+ and initiate lipid peroxidation will form v i a catalase-insensitive autoxidation of the released Fe 2+, which is favored by A D P included to facilitate iron release. p e r h a p s via disruption of the t e r n a r y complex of ironbinding proteins (34}, and also to chelate the released iron (33). However, excess A D P will cause the autoxidation of too m u c h Fe 2+, thereby shifting the Fe2+/Fe 3+ ratio toward values t h a t are not permissive to lipid peroxidation. As a result of the equilibrium between Fe 2+ release and Fe 2+ autoxidation, Saito e t al. (34) have shown t h a t xanthine oxidase and transferrin dependent lipid peroxidation is enhanced by low concentrations of A D P b u t inhibited by high concentrations of this s a m e chelator, irrespective of the presence or absence of catalase to remove H202 (34). I t has been suggested t h a t 02" dependent Fe 3+ reduction and lipid peroxidation can also be achieved by replacing xanthine oxidase with "autoxidizable" compounds like thiols and ascorbate (35,36}. However, others have shown t h a t "autoxidation" of thiols and ascorbate at the expense of molecular oxygen is neither t h e r m o d y n a m i c a l l y or kinetically feasible unless iron is present to serve as direct one electron acceptor (1,18,27,37). Thus, ascorbate or thiol oxidation is mediated by direct electron addition to Fe 3+. Subsequent reactions of Fe 2+ with oxygen can eventually form O~'. In agreement with this interpretation, Table 6 indicates t h a t replacing x a n t h i n e oxidase w i t h glutathione or ascorbate resulted in SOD-insensitive lipid peroxidation, indicative of 02" independent ADP/Fe 3+ reductiom Moreover, results presented in Table 6 indicate t h a t catalase did not inhibit ascorbate or glutathione dependent lipid peroxidation, perhaps indicating either t h a t ADP/Fe 2+ autoxidized so slowly t h a t H202 could not accumulate to oxidize more Fe 2+ and prevent initiation; or t h a t H202 was formed in such a way t h a t it could not be intercepted by catalasr Overall, it seems t h a t lipid peroxidation is mediated b y ADP/Fe 8+ reduction and t h a t ADP/Fe 2+ would in turn combine with residual ADP/Fe 3+ to form some t y p e of Fe2+/Fe 3+ complex. A s c o r b a t e is an effective iron reductant, therefore relatively minor changes in its concentration will result in substantial changes in the Fe2+/Fe 3+ ratio and lipid peroxidation. For example, 25 tdVl ("low") ascorbate reduces some Fe 3+ to Fe 2+ and promotes lipid peroxidation, whereas 50 ~M ("high") ascorbate reduces too m u c h LIPIDS, Vol. 27, no. 3 (1992)

224

REVIEW TABLE 7

Ascorbate-Dependent Lipid Peroxidationa, b System

nmol MDA/mL/min

"Low" ascorbate + H202 "High" ascorbate + H202

0.77 0.60 0.16 0.92

% Change -22 +475

a Incubationscontainedmicrosomalphospholipidliposomes(1 ~mol lipid phosphate/mL) and ADP/Fe3+ (250 ~M-50 ~M) in 50 mM NaCI, pH 7.0, 37~ Reactionswere started by addition of either "low" (25 ~Vl)or "high" (50 ~M) ascorbate and lipid peroxidation was assayed as malondialdehyde(MDA)formation (3). Where indicated, 50 ~M H202 was also includedin the reaction mixtures. bData from Miller and Aust (18).

Fe3+ to Fe 2+ and inhibits lipid peroxidation (Table 7). Very similar observations have been made with thiols like cysteine or dithiothreitol, which also promote moderate to excessive iron reduction upon minor increase in their concentration {38). Glutathione is a much weaker reductant than is ascorbate, cysteine or dithiothreitol (cf refs. 18,38). Therefore, glutathione may not achieve the complete reduction of iron and inhibition of lipid peroxidation, even when it is used at rather high concentrations. Not surprisingly, both glutathione and ascorbate were capable of promoting lipid peroxidation, yet the concentration of glutathione was approximately 80-fold that of ascorbate (cf legend to Table 6). From another viewpoint, concentrations of glutathione favoring lipid peroxidation were 20-fold those of ascorbate that inhibited lipid peroxidation (cf Tables 6 and 7). One can use H202 to modulate the balance of reduced vs oxidized iron and either inhibit or stimulate lipid peroxidation. For example, with "low" concentration of ascorbate and partial Fe3+ reduction, the addition of H202 will reconvert more Fe 2+ to Fe3+, thereby inhibiting lipid peroxidation {Table 7). With "high" concentration of ascorbate and extensive or eventually complete Fez+ reduction, the addition of H20= will oxidize some Fe 2+ to Fe 3+, thereby stimulating lipid peroxidation (Table 7). These experiments indicate that reductants/oxidants can facilitate or prevent lipid peroxidation by virtue of their ability to modulate the Fe2+/ Fe3+ ratia In particular, the effects of H202 on ascorbate dependent lipid peroxidation indicate that its ability to stimulate or inhibit will solely depend on how much iron is maintained in the Fe z+ form to achieve the formation of appropriate Fe2+/Fe3+ ratios. POSSIBLE EXPLANATIONS FOR THE REQUIREMENT FOR BOTH FE 2+ AND FE 3+

The results described thus far indicate that the simultaneous availability of Fe2+ and Fe3+ determines whether lipid peroxidation will occur. The diverging effects of ~ scavengers and the observations that SOD and catalase can either stimulate, inhibit or not affect lipid peroxidation apparently rule out any major role for ~ in initiation of lipid peroxidation. These and other considerations, such as the previously mentioned diffusion-limited reactivity of "OH, have led Bucher et aL (11), Minotti and Aust LIPIDS, Vol. 27, no. 3 (1992)

(15,16,27} and Braughler et aL (12-14) to propose that lipid peroxidation is mediated by some type of iron/oxygen complex comprised of both Fe 2+ and Fe 3+, and perhaps oxyger~ All attempts by our laboratories to characterize this elusive complex have been unsuccessful so far. But studies of Co2+ or Cos+ dependent reactions have shown that the formation of oxygen-bridged complexes of reduced and oxidized metals is feasible {39). Other investigators (40,41} have proposed that the iron/oxygen complex involved in lipid peroxidation can be best described as the perferryl ion (Fe3+O~'), which forms via the following reactions of Fe 2+ with oxygen, or the reduction of Fe 3+ with 02". F e 2+ -Jff 0 2 ~ Fe2+O2 ~ F e 3+ -~- O ; "

[14]

Interestingly, Goddard and Sweeney (42) and Ursini et aL (43) have noted that the requirement for both Fe 2+ and Fez+ may underlie the initiation by perferryl iron rather than by ferrous/dioxygen/ferriccomplex(es) for at least two reasons. First, perferryl iron formation and reaction with lipids best occur with chelators and chelator/Fe2+ ratios that favor only moderate Fe 2+ autoxidation and generate an equilibrium between Fe2+O2 and Fe3+O~" (43). Second, excess Fe2+ would compete with lipids as electron donors for Fe3+O~", thereby inhibiting hydrogen abstraction and lipid peroxidation (42,43). This proposed competition between lipids and excess Fe2+for perferryl ion seems consistent with our findings that lipid peroxidation is maximal when critical amounts of Fe 2+ oxidize to Fe 3+ (Tables 3-5}. Therefore, the perferryl ion model of lipid peroxidation is intriguing and deserves experimental attentiorL However, excellent rates of lipid peroxidation have been observed in incubations containing H202 and unchelated Fe 2+, which may not autoxidize to generate the equilibrium of Fe2+O2 with Fe3+O~" (Tables 1 and 2). Ftm thermore, SOD did not inhibit lipid peroxidation induced by Fe 3+ plus ascorbate or glutathione (Table 6) nor that induced by Fe 2+ chelates (11,16) perhaps indicating that an 02"-centered radical like perferryl ion may not be involved in either of these systems. Xanthine oxidase dependent lipid peroxidation is inhibited by SOD (Table 5) because Fe 3+ reduction is mediated by O~" in this system, regardless of whether Fe 2+ is subsequently utilized to form the perferryl ion or a ferrous/dioxygen/ferric complex. Overall, there seem to be some positive but also some negative considerations to support the initiation by perferryl ion rather than by a ferrous/dioxygen/fe~ ric complex. Schaich and Borg (8) have proposed that irrespective of the nature of the reactive species which abstracts hydrogen from lipids, excess Fe 2+ would compete as electron donors for LOO ~ and LO', thereby inhibiting both the formation of first LOOH and subsequent LOOH dependent propagation. As an additional criticism, it has been emphasized that most lipid peroxidation systems rely on high concentrations of iron, which not only fail to reproduce in vivo situations but also exaggerate the "antioxidant" effects by Fe 2+ (8). The validity of this criticism is somehow weakened by previous observations that the requirement for both Fe 2+ and Fe3+ is evident in ferritin and transferrin dependent systems, i.e., systems mediated by physiological iron storage proteins via the reductive mobilization and subsequent autoxidation of

225

REVIEW very low a m o u n t s of Fe 2+ (33,34}. We therefore conclude t h a t Fe 2+ oxidation is required not to decrease the concentration of Fe 2+ as an antioxidant, but rather to increase the concentration of some type of oxidant t h a t requires Fe 3+. Similar observations have recently been made for the initiation of lipid peroxidation by nonheme iron embedded in the microsomal milieu (20}. However, lack of information on the precise structure of microsome~ bound iron does not justify extensive discussion of these latter observations in the present article. The importance of attaining an optimal Fe2+/Fe 3+ ratio in the initiation of lipid peroxidation was originally proposed and reiterated by relatively few investigators (11-16,27). A n overview of the recent literature, however, reveals t h a t similar results have been obtained by other research groups (44-47). Nevertheless, the mechanism of lipid peroxidation remains a m a t t e r of controversy. For example~ it is certainly true t h a t lipid peroxidation can be promoted by the direct addition of appropriate a m o u n t s of Fe 2+ and Fe ~+ yet A r u o m a et al. (28) have found t h a t Pb 2+ and AP + can replace Fe 8+ in the initiation of lipid peroxidation. This latter finding has been taken as evidence against the initiation of lipid peroxidation by a specific Fe 2+/Fe 3+ complex. However, these investigators did not investigate the effects of Pb 2+ or AP + on Fe 2+ oxidation or the subsequent Fe2+/Fe 3+ ratio. This report also did not address the usual hypothesis t h a t the "OH was responsible for lipid peroxidation. I t should be noted, however, that Pb 2+ and AP + have also been found to inhibit the lipid peroxidation promoted by ADP/Fe 3+ and either ascorbate or the reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent microsomal electron transport system, and t h a t the reasons for such discrepancies have remained unexplained (28). Thus, the participation of A P + and Pb 2+ in iron-dependent lipid peroxidation remains confined in very narrowed experimental conditions, whereas the formation of some type of Fe2+/Fe ~+ complex appears to mediate the initiation of lipid peroxidation in several model systems. We believe t h a t the identification of intracellular low molecular weight iron complexes and the characterization of the mechanisms by which the cell regulates the redox state of these complexes and perhaps their interaction with other metals remain absolute prerequisites to further investigations in this field. In conclusion, we have presented evidence t h a t lipid peroxidation is mediated by some type of Fe2+/Fea+ complex which can be formed by Fe a+ reduction or by Fe 2+ oxidation, and t h a t these latter reactions can occur and be affected by various mechanisms.

ACKNOWLEDGMENTS This research was supported in part by NIH grant number ES05056. We thank Timothy P. Ryan for helpful suggestions and Terri Maughan for assistance in preparation of this manuscript.

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[Received June 13, 1990, and in revised form March 18, 1991; Revision accepted April 20, 1991]