ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 297, No. 2, September, pp. 258-264, 1992

Inhibition of Lipid Peroxidation by Iron(lll) and Ascorbate

Promoted

Dorothy C. Beach and Eugene Giroux’ Marion Merrell Dow Research Institute,

Received February

2110 E. Galbraith Road, Cincinnati,

20,1992, and in revised form May 12,1992

Peroxidation of rat liver microsomes and of phospholipid isolated from them was studied using iron(II1) and ascorbate initiation. One-half equivalent of citrate per iron equivalent maintained solubility of the metal ion at neutral pH. Several metal chelators, including additional citrate, blocked peroxidation, but catalase did not. These characteristics are consistent with those reported by others (D. M. Miller and S. D. Aust (1989) Arch. Biochem. Biophys. 271, 113-119). Several antioxidants, principally tocopherol analogues and nitroxides, and, as well, a nonenzymatic component of “thymol-free” catalase, potently blocked lipid peroxidation, or, equivalently, dioxygen depletion from suspensions of peroxidizing microsomes. Chromanols were the most active antioxidants. No thiol studied had significant antioxidant activity in 0 1992 Academic Press, Inc. the test system.

Tissue injury upon reperfusion of an ischemic organ has been ascribed in part to generation of oxygen-derived radicals (1). The three-electron reduction product of dioxygen is particularly reactive. Generation of hydroxyl radical by Fenton’s reaction requires hydrogen peroxide and a transition metal. Most common in typical biological milieu are iron and copper. Oxygen radical-mediated lipid peroxidation requires a transition metal (2), although there are in vitro models in which the peroxidation process does not involve hydroxyl radical (3). We studied peroxidation of rat liver microsomes and of phospholipid material further fractionated from microsomes following addition of iron(II1) and ascorbic acid in the presence of dissolved dioxygen with the goal of determining classes of agents that blocked peroxidation with some potency. Effects of chromanols, alkylated phenols, nitrones, nitroxides, and thiols in various peroxidation models have been reported, but we are not aware of comparisons of potency of these antioxidants in a single system. A variety 1 To whom correspondence 258

Ohio 45215

should be addressed. Fax: (513) 948-6439.

of systems have been investigated in modeling in vivo radical processes. We wished to study a metal-dependent process and preferred an iron-based model over a copperbased model. Hydroxyl radical generation catalyzed by the latter metal typically is blocked by nonspecific protein interference (4), which would seem to lessen its general physiological relevance. Iron(II1) was preferred over iron(I1) because of the instability of the divalent cation at neutral pH. Both a complexant, to keep Fe(II1) in solution, and a reductant, to initiate peroxidation, were needed. EDTA as complexant specifically has been called a major factor influencing a multiplicity of mechanisms in iron-dependent peroxidation (5) and so we chose to use citrate, as this molecule may be one physiological ligand of the small amount of iron which is not proteinbound (6). A system based on iron, citrate, and ascorbate, described by Miller and Aust (7), is most similar to the one studied here. In that model, lipid peroxidation did not involve hydroxyl radical and a Fe(II):Fe(III) complex, which probably requires dioxygen, was proposed as an active species (7). In an iron, EDTA, and ascorbate system described by Klein et al. (8), hydroxyl radical was shown to participate in oxidation of dimethyl sulfoxide to formaldehyde. Factors responsible for all of the differing features of the several model iron-based systems have not been unequivocally sorted out, but it has been suggested (3) that susceptibility of lipids to peroxidation by mixed-valence iron complexes may not be shared by other oxidizable substrates and that iron complexants are a dominant factor determining reaction mechanism. It is also unclear which features may be most germane in a physiological context. We demonstrated that catalase, at much greater than catalytic concentrations, was ineffective in blocking peroxidation, indicating that hydrogen peroxide (and, subsequently, hydroxyl radical) was not an intermediate. Nonenzymatic component(s) of the commercial catalase preparation did block peroxidation and this observation was further explored. All iron-complexing agents tested, including additional citrate, inhibited peroxidation, a re0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resf~~ed.

INHIBITION

OF IRON-BASED

sult also inconsistent with participation of hydroxyl radical (9-11). Several antioxidants (tocopherol analogues, TEMP02, thymol) blocked peroxidation. The rate of dioxygen depletion monitored blockade of peroxidation, as did the rate of TBARS formation. The most potent nitroxide antioxidants were slightly less active than potent chromanol antioxidants. Such studies may uncover antioxidants to test in other systems and may aid in elucidating mechanisms of initiation and propagation of peroxidation in the iron-ascorbate system. MATERIALS

AND METHODS

Protein was determined by the Lowry method, as detailed by Peterson (12). Wet ashing of lipid and phosphate determination were carried out using standard procedures (13, 14). Wet ashing of microsomes prior to atomic absorption spectroscopy used nitric acidperchloric acid (4:l). Unless otherwise mentioned, all preparative procedures were carried out at 4°C and all assays were carried out at 37°C. Water was processed with a Mini-Q system (Millipore) before use. The MDL-denominated chromanol derivatives were obtained from J. M. Grisar (15). The Captopril sample and the other MDL-numbered compounds were synthesized in this research center. Most antioxidants were purchased from Aldrich Chemical. Desferoxamine was from Ciba-Geigy. Other chemicals were reagent grade. Microsomes were prepared from fresh rat liver by differential centrifugation of homogenates in buffered 0.25 M sucrose (16). The final pellet was resuspended in a volume of 125 mM KC1 equal to the original wet liver weight. Aliquots were stored at -20°C. Phospholipids were obtained from freshly prepared microsomes by Folch extraction (17). All solvents were argon-sparged. Lipid material after rotary evaporation of solvent was solubilized with chloroform:methanol (2:1), using about 1 ml per 5 g original wet liver weight. Argon-gassed aliquots were stored at -20°C. Peroxidation trials were carried out in a buffer of 0.1 M NaCI, 10 mM sodium cacodylate, 0.1% polyethylene glycol6000, pH 7.4. Phospholipid in chloroform/methanol was concentrated under a stream of argon and taken up in ethanol; 1 vol of this was dispersed by vortexing into 99 vol of buffer warmed to assay temperature. Microsomes were dispersed in buffer. Iron(II1) stock (50 mM) was prepared by dissolving Fe&SO& in buffer, with heating. Trisodium citrate and sodium carbonate, both 25 mM, were added to the warm solution, thus the molar stoichiometry of Fe(III):citrate was 2:l. At room temperature, the solution was adjusted to pH 7.4 with solid sodium carbonate, microfiltered, and protected from light. This procedure avoided precipitation of polymeric iron species (18). Iron concentration was verified by atomic absorption spectroscopy. Ascorbic acid was dissolved at 50 mM concentration in argon-sparged buffer before each experiment. To quantify peroxidation, an aliquot of 0.5 ml of assay mixture was mixed with 1 ml of 10% trichloroacetic acid (w/v) containing 0.02% butylated hydroxytoluene. To this was added 1.5 ml 0.67% thiobarbituric acid. After heating in a boiling water bath for 20 min, then cooling, then clarification by low-speed centrifugation, absorbance at 532 nm was measured (19). The result (TBARS) was referred to a standard curve of malondialdehyde equivalents constructed using up to 20-nmol amounts of tetraethoxypropane. The measured millimolar extinction coefficient was 159. In trials of agents blocking peroxidation, test mixtures were sampled at l-min intervals for 6 or 7 min. Within the precision of the assays, data could be fit with straight lines and slope values were used to characterize initial rates of formation

’ Abbreviations used: Captopril, D-3-mercapto-2-methylpropanoyl-lproline; MDA, malondialdehyde; TBARS, thiobarbituric acid reactive substances; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; Trolox, 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

259

PEROXIDATION

of TBARS. Concentrations of added iron(II1) and ascorbate in antioxidant trials were consistently 25 pM each. As representative data, in 12 trials over a 5-week period using 0.33 mg protein per ml of a single microsome preparation, the mean rate of peroxidation was 0.34 + 0.02 (SEM) nmol MDA per minute per 0.5 ml. In a series of 9 trials over a B-month period using 0.5 pmol phosphate per ml of a single lipid extract, the mean rate was 0.74 i 0.02 (SEM) nmol MDA per minute per 0.5 ml. Na+,K+-ATPase was prepared from dog outer renal medulla (20,21) at a specific activity of 20 units/mg protein. Before use in peroxidation trials, enzyme was diluted to a convenient volume with cold buffer and pelleted by ultracentrifugation. Material resuspended in buffer was added to test systems after addition of iron hut before addition of ascorbate. Catalase was assayed (22,23) using a Clark oxygen electrode at 30°C in pH 7.0 buffer (50 mM sodium, potassium phosphate, 1 mM diethylenetriaminepentaacetic acid, 0.1% Triton X-100). Bovine liver catalase was purchased from Sigma; this material (item C-40, thymol added during purification was subsequently removed by dialysis, according to Sigma, so the material was labeled thymol-free) was 60% protein by weight. A 10 mg/ml solution was dialyzed at room temperature against assay buffer containing 5 mM 3-amino-1,2,4-triazole and 5 mM hydrogen peroxide to irreversibly inactivate the enzyme (24,25). Enzyme solution was subsequently frozen, lyophilized, reconstituted to a small volume, and chromatographed on a 0.9 X 29-cm column of Sephacryl S-200, equilibrated and eluted with a mixture of 9 parts peroxidation buffer, 1 part glycerol. The protein peak eluting from the column was 40% of the mass of the starting material. Another portion of catalase was carried through the protocol except dialysis was against buffer, which did not contain the irreversible inhibitor. A third portion of catalase was subjected only to solvent-exchange chromatography. Specific activities of inactivated material and its process control material were 18 and 81%, respectively, of the specific activity of the minimally processed portion. In addition, a solution of Sigma catalase, 1 mg solid per milliliter buffer, was heated from room temperature to 70°C at 1°C per minute. This was 10°C beyond the temperature at which a pilot experiment indicated catalase lost over half its original activity. Coagulated enzyme was removed by centrifugation. Supernate had no detectable enzyme activity, under conditions where 1% of the activity of unheated solution was easily measurable. A representative microsome preparation from rat liver contained catalase activity equivalent to 0.5 mg of bovine enzyme per 100 mg protein. Dispersions of phospholipid extracted from microsomes, at concentrations used in peroxidation trials, did not contain measurable catalatic activity. Clark electrode measurements were made with a Yellow Springs Instruments Model 5300 biological oxygen monitor. Voltage at the recorder output was sampled every few seconds by an analog-to-digital conversion board linked to a personal computer and stored for subsequent analysis. The rate of change in oxygen content most often used to characterize an assay was taken as the initial slope value of a low-order power series fit by least-squares regression. Sigmoid curves describing blockade of peroxidation were characterized using the ALLFIT program (26).

RESULTS Characterization

of Peroxidation

System

Peroxidation of rat liver microsomal lipids required the presence of each of the following reactants: lipid substrate, iron(III), ascorbate, and dioxygen. Orienting experiments indicated convenient concentrations for peroxidation trials in the range of 0.25-0.5 mg microsomal protein per milliliter or 0.25-0.5 pmol dispersed lipid phosphate per milliliter and 25 ~~-100 PM ascorbate and iron concentrations. For convenience, equal concentrations of iron(II1) and ascorbate were used. Iron concentrations in

260

BEACH

AND

six microsome preparations were determined by atomic absorption spectroscopy. A typical peroxidation trial with 0.25 mg microsome protein per milliliter was estimated to contain 5 PM iron added as a component of the microsome material. Tests with microsomes and ascorbate but no added Fe(II1) yielded TBARS response consonant with such a level of contaminating transition metal. The iron content of Folch extracts of microsomes or of buffer solutions was not determined. Conclusions drawn from tests using microsomes or phospholipid dispersions were consistent with one another. Which of the two substrates was used is indicated for each experiment below. At 50 PM concentrations of added iron(II1) and ascorbate, generation of TBARS in a phospholipid dispersion ceased within 10 min. Such an exhausted system was renewed by supplementation with ascorbate. Additional ascorbate, equal to the original amount, approximately duplicated the original effect in the initial rate of TBARS formation (about 70% of the original) and in the extent of TBARS formed after 10 min (about 80% of the original). Blocking of Lipid Peroxidation Metal-complexing agents blocked lipid peroxidation in microsomes (and dioxygen depletion, as illustrated for TEMPO, below). Desferoxamine, diethylenetriaminepentaacetic acid, and EDTA, 50 PM in trials using 25 PM iron(III), blocked completely. Nitrilotriacetic acid, at similar stoichiometry, blocked by 70%. Sodium citrate, 50 j.&M, added to a mixture already containing 12.5 PM citrate and 25 PM Fe(II1) lowered the rate of TBARS formation to half the control value. Bovine liver catalase, irreversibly inactivated to 18% of the specific activity of a reference sample, was without effect on the course of lipid peroxidation. In control mixtures containing 0.5 pmol microsomal lipid phosphate per milliliter, 25 PM each Fe(II1) and ascorbate, peroxidation occurred at a linear rate over ‘I-min assays at 0.61 nmol MDA per minute per 0.5 ml. Peroxidation occurred in mixtures containing 0.18 and 0.24 mg inactivated catalase protein/ml at rates of 0.65 and 0.63 nmol MDA/min/0.5 ml, respectively. Another catalase sample, subjected to similar preparative manipulations except for exposure to irreversible inhibitor, had 81% of the specific activity of the reference material. In mixtures containing 0.14 and 0.19 mg of the catalase protein per milliliter peroxidation occurred at rates of 0.61 and 0.57 nmol MDA/min/0.5 ml, respectively. None of these peroxidation rates in the presence of catalase was significantly different from the control value. These tests were carried out using dispersions of phospholipid prepared by organic solvent extraction of microsomes; such dispersions had no endogenous catalatic activity. Concentrations of added enzyme were several orders of magnitude greater than those needed to demonstrate catalatic activity. In contrast, a

GIROUX

sample of catalase chromatographed only to effect solvent exchange did block peroxidation in phospholipid dispersions. The I&,,, for this material was 0.03 mg protein per milliliter. Enzyme-free supernate obtained by heating a solution of Sigma catalase also blocked peroxidation in microsomes with an IC& value equivalent to 0.07 mg initial solid per milliliter. This suggested by a second approach that peroxidation blocking activity in the commercial material was not catalase. Thymol, of which this material was supposedly free, blocked peroxidation in microsomes with an ICs,, of 0.05 mM; thymol content of the material purchased from Sigma would have to be several percent to account for the observed antioxidant effect. Complete blockade of peroxidation could be achieved by MDL 73,404, a cationic chromanol derivative. The I&,, value was estimated as 2.6 PM in phospholipid dispersions. With microsomes as substrate, the I&,,, value of MDL 73,404 was estimated to be 3.4 PM (Fig. 1). The rate of oxygen consumption in suspensions of microsomes undergoing peroxidation could also be used to assess antioxidant agents. TEMPO reduced the rate of oxygen depletion in proportion to its concentration in the test mixture (Fig. 2). This figure illustrated better than sparse discrete-point TBARS data that the effect of antioxidant was continuous over the observation interval. Inhibition by TEMPO of microsomal peroxidation as assessed by TBARS formation and by oxygen consumption is illustrated in Fig. 3. By TBARS and dioxygen depletion methods, the I&,,, values were calculated to be 14 and 13 j.&M, respectively. A few trials were carried out with Trolox and MDL 73,404 to establish the general applicability of this use of the Clark electrode. Results obtained for these compounds were completely consistent with those obtained by monitoring inhibition of TBARS formation.

10 [Antioxidant] (PM) FIG. 1. Rates of TBARS formation were measured in suspensions of rat liver microsomes (0.33 mg protein/ml) containing 25 pM concentrations of added iron(II1) and ascorbate. Aliquots were taken for color development each minute for 7 min for control mixtures and mixtures containing various concentrations of MDL 73,404 (circles) or Trolox (squares). Rates were expressed as percentages of the inhibitor-free control rate.

INHIBITION

OF IRON-BASED

0.6--

Time (min) FIG. 2. Dioxygen depletion in suspensions of rat liver microsomes (0.25 mg protein per ml) following addition of 25 pM Fe(II1) and ascorbate (at Time 0). TEMPO concentration was 0 (control, open square), 13 pM (filled square), or 27 pM (diamond).

Several classes of potential blocking agents were tested for effects on peroxidation of microsome suspensions. I&,, values were determined only for compounds active below 0.1 mM concentration. Results are presented in Table I. Chromanols, nitroxides, and antioxidant phenols demonstrated varying degrees of potency. No aliphatic or aromatic thiol listed in the table significantly blocked peroxidation at the highest concentration tested. Lipid peroxidation in assay mixtures containing 0.5 pmol lipid P per milliliter, 25 PM concentrations of iron(II1) and ascorbate, with 15 or 30 pg/ml concentrations of dog kidney Na+,K+-ATPase were 0.62 and 0.65 nmol MDA per minute per 0.5 ml, respectively, compared to the ATPase-free control rate of 0.64 nmol MDA per minute per 0.5 ml. These two ATPase concentrations, and others tested as well, were without significant effect on peroxidation. DISCUSSION The present experimentation examined blockade of lipid peroxidation in a metal-dependent test system by several classes of agents. The model used was similar to one of Miller and Aust (7), but differed in details such as buffering and proportions of iron:citrate and iron:ascorbate. Ascorbate consumption was responsible for cessation of TBARS formation after a few minutes in this system. In an iron(hydrogen peroxide peroxidation system, it was additional Fe2+ that reinitiated oxygen consumption (27). An agent that would deplete a mixture of one of the reactants would block peroxidation. Also, any agent that competed with lipid substrate for participation in reactions involving iron(III), ascorbate, and.dissolved oxygen would block TBARS formation. If peroxidation of lipid in this system is envisaged as a multistep sequence beginning with initial hydrogen atom abstraction, followed by reaction of dioxygen with a radical intermediate and

261

PEROXIDATION

further reactions (28), an agent could be an antioxidant by reversing or blocking any step before those generating species measured as TBARS. Where tested, active compounds in Table I inhibited TBARS and dioxygen depletion with equal potency. Blockade of peroxidation by metal chelators was not limited to desferoxamine and diethylenetriaminepentaacetic acid. EDTA, nitrilotriacetic acid, and citrate, in addition to the two other chelators, blocked peroxidation in an order of potency similar to their avidity for iron complex formation. The 5:l citrate:Fe(III) ratio used by Miller and Aust (7) was lo-fold higher than the one we used and we estimate that peroxidation was severalfold lower in their system due to this excess of chelator. Some complexant is needed to keep iron(II1) in solution at neutral pH, but we conclude from observations of Spiro et al. (18) that little or none of the 25-100 PM iron in our peroxidation system was present as polymeric iron species. The choice of citrate:iron ratio was made on the basis of oxygen electrode measurements of iron(III)-ascorbate solutions where 1:l citrate:iron clearly slowed oxygen consumption relative to mixtures of 0.5:1 stoichiometry. Low concentrations of iron(II1) and ascorbate and monitoring only the early portion of the peroxidation process were chosen in order to evaluate modest concentrations of compounds capable of antioxidant activity. Several classes of compounds besides metal chelators blocked peroxidation at concentrations below the evaluation limit of 100 PM. MDL 73,335 and MDL 73,404 (see footnote to Table I for systematic names) were the most potent agents. These tocopherol analogues prevent spontaneous lipid autoxidation in rat brain homogenates and reduce infarct size in myocardial infarct/reperfusion studies in rats (15). Both Trolox and MDL 73,362 have a water-solubilizing carboxylate. The superior antioxidant 100

T

[TEMPO1(PM) FIG. 3. Blockade by TEMPO of TBARS formation (filled squares) and of dioxygen depletion (open squares) in suspensions of rat liver microsomes (0.33 and 0.25 mg protein/ml for the two types of trials, respectively), provoked to undergo lipid peroxidation by addition of 25 pM iron(II1) and 25 pM ascorbate. Rates were expressed as percentages of the inhibitor-free control rates.

262

BEACH

AND TABLE

Inhibition Antioxidant Chromanols

I&o f SE (PM)

TEMPO’ DOXYL-cyclohexane” 4-Hydroxy-TEMPO’ 3-Carboxyl-PROXYL’ N-t-butylphenylnitronec MDL 6,533” MDL 101,002’

I Lipid

Peroxidation Antioxidant

and similar compounds

MDL 73,335” MDL 73,404” Trolox” MDL 73,362d Nitroxides

of Microsomal

GIROUX

IC, k SE (FM) Phenols

1.3 3.4 54 90

i 0.1 2 0.2 f 4 +10

Thymol ProbucoP MDL 29,311h

and similar compounds

48

* 4 N.A.* N.A.*

Thiols 14 48 95

r!Y 1 f 5 fll N.A. N.A. N.A. N.A.

Captopril N-(2-Mercaptopropionyl) MDL 19,327’ MDL 27,955) MDL 28,365’ MDL 28,963’ MDL 29,591m MDL 29,724” MDL 29.752’

N.A. glycine N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.

Note. Suspensions of rat liver microsomes were provoked to undergo peroxidation by addition of 25 pM iron(II1) (with 12.5 pM citrate) and 25 Potential antioxidants were evaluated at 100 pM; if this concentration did not reduce the rate of TBARS formation below half the control value, they were considered not active (N.A.). Measurements of blockade of oxygen consumption were incorporated with TBARS formation rates in some I& calculations: 2 of 11 for MDL 73,335 and Trolox; 2 of 18 for MDL 73,404; 10 of 19 for TEMPO; 4 of 9 for DOXYL-cyclohexane and 4-hydroxyTEMP0. TBARS data only (9 measurements each) were used in I&a calculations for MDL 73,362 and thymol. ’ 3,4-dihydro-2-(2-dimethylaminoethyl)-2,5,7,8 tetramethyl-2H-1-benzopyran+ol * 3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-l-benzopyran-2-ethanaminium 4-methylbenzenesulfonate ’ Aldrich Chemical Co. d 2,3-dihydro-5-hydroxy-2,2,4,6,7-pentamethyl-3-benzofurancarboxylic acid e 5,6-dihydro-6,6-dimethyl-2-phenylpyrazin-3(4H)-one l-oxide ‘3,4-dihydro-3,3-dimethylisoquinoline, 2-oxide g 4,4’-(isopropylidenedithio)bis(2,6-di-t-butylphenol) h 4,4’-[methylenebis(thio)Jbis[2,6-bis(l,l-dimethylethyl)phenol] ’ 2,3-dihydro-N,N,5-trimethyl-2-thioxo-lH-imidazole-4-carboxamide j (2,3-dihydro-5-methyl-2-thioxo-lH-imidazol-4-yl)[4-(methylthio)phenyl]methanone k (5-ethyl-2,3-dihydro-2-thioxo-lH-imidazol-4-yl)-4-pyridinylmethanone ’ [3,4-bis(methylthio)pheny1](2,3-dihydro-5-methyl-2-thioxo-1H-imidazol-4-yl)methanone m l-ethyl-1,3-dihydro-5-ethyl-4-(4-pyridinyl)-2H-imidazole-2-thione ” 1,2-dihydro-6-methyl-5-(4-pyridinylcarbonyl)-2-thioxo-3-pyridinecarbonitrile ’ l-butyl-1,3-dihydro-5-methyl-4-(4-pyridinyl)-2H-imidazole-2-thione * Probucol and MDL 29,311 were not active in the typical trial, most likely due to insolubility. When mixed with phospholipid in ethanol before preparation of lipid dispersion in aqueous assay medium, each had a potent antiperoxidative activity. pM ascorbate.

activity of similar compounds lacking the acid functionality has been rationalized on the basis of stereoelectronic effects (29). The three active nitroxides in Table I have been shown to maintain viability of cells exposed to the superoxide-initiated oxygen free-radical cascade (30) and TEMPO has protective effects in two rat models of cardiac ischemia/reperfusion injury (31). Aliphatic thiols Captopril and N-(2-mercaptopropionyl)glycine were tested because they ameliorate postischemic myocardial dysfunction in rabbit (32). The aromatic thiols were selected because of their high reactivity with the stable free radical l,l-diphenyl-2-picrylhydrazyl(33). None of the thiols potently blocked TBARS formation below our arbitrary test concentration of 0.1 mM. Cysteine blocked oxidation with varied efficacy in two azo decomposition-initiated systems

(34). Na+,K+-ATPase is a thiol-rich enzyme and possibly is a target of radical inactivation in vivo; when incorporated into liposomes it is inactivated during lipid peroxidation initiated by the Fenton reaction (35). We found the enzyme did not diminish TBARS formation in our phospholipid dispersion test system. It appears that mechanism(s) by which thiols influence ischemia/reperfusion injury cannot be modeled with this peroxidation system. Liebler et al. (27) noted a lag in the maximum rate of dioxygen consumption in suspensions of soybean phosphatidylcholine liposomes oxidatively challenged with Fe’+ and H,O,; the lag was increased in proportion to the amount of a-tocopherol incorporated into liposomes. Scheschonka et al. (36) noted a marked lag in TBARS

INHIBITION

OF IRON-BASED

accumulation in vitamin E-protected microsomes incubated with ADP-Fe(II1) and ascorbate. Effects of cysteine and vitamin E on dioxygen depletion from suspensions of oxidizing soybean phosphatidylcholine liposomes illustrated by Motoyama et al. (34) included partial blockade early on, succeeded by depletion at a more rapid rate; the effect most responsive to antioxidant concentration under most conditions was the duration of early inhibition. We noted no lags in TBARS formation or in oxygen consumption in either unprotected or antioxidant-protected microsome suspensions. Inhibition of initial rate using either measured parameter was proportional to the concentration of blocking agent. This group of characteristics is most similar to those of an aqueous phase scavenger (cysteine) on oxidations initiated in a lipid (liposome) phase, as described (34). Ascorbic acid is known to regenerate a-tocopherol from its sacrificial oxidative products, prolonging antioxidative effect (27,37). Ascorbic acid may have had antioxidant-sparing as well as prooxidant activity in microsome suspensions protected with active compounds in Table I. Catalase has been used in many in vitro and in vivo studies to demonstrate participation of hydrogen peroxide and, by implication, hydroxyl radical, in processes under investigation. The demonstration is most believable when it is shown that observed effects are due uniquely to the catalatic activity of the enzyme and catalytic concentrations of enzyme should suffice. In systems of known volume, this would not be greater than pg/ml concentrations (22, 25). Neither catalase nor inhibited enzyme of onefourth the activity blocked peroxidation in our trials using organic solvent-extracted phospholipids. The microsome preparation contained easily measurable catalatic activity, yet, despite this, peroxidation did occur. Noncatalatic component(s) of the commercial enzyme preparation blocked peroxidation. Antioxidant activity was found in supernate from heat-coagulated enzyme solution and we speculate that in the batch of “thymol-free” enzyme we used, thymol was incompletely removed, since thymol was an effective antioxidant (Table I). The possibility of antioxidants in other batches merits consideration by purchasers of catalase, since two of five commercial catalase preparations were found to inhibit microsomal lipid peroxidation (38). Thus, hydrogen peroxide susceptible to dismutation by catalase was not a significant intermediate in the peroxidative sequence, ruling out hydroxyl radical as a major initiator of peroxidation in this system, consistent with observations of Miller and Aust (7). These authors used Sigma thymol-free catalase, but if their material had contained antioxidant equivalent to the batch we used, they would not have seen effects of contaminant at the microgram amounts employed. In vivo experiments may employ greater amounts of thymol-free catalase [for example, (32)], since concentrations at critical sites will not be known. In such a situation, effects ascribed to catalase may be confounded by antioxidant contaminant.

PEROXIDATION

263

Minotti and Aust (3) have generalized that which among several reaction pathways predominate in ironpromoted lipid peroxidation depends strikingly on the iron complexant used in the system, and furthermore that lipid peroxidation should not be considered equivalent to other radical-scavenging systems. Among the variety of model systems employed to isolate key features of more complex systems, discordant conclusions might be rationalized on such bases. In the biological environment, many iron ligands and many substrates susceptible to radical attack likely are present. The degree to which positive and negative results obtained in the model system studied here may predict results in more complex systems must be established by further investigation. REFERENCES 1. McCord, J. M. (1985) N. Engl. J. Med. 312, 159-163. 2. Gutteridge, J. M. C., Richmond, R., and Halliwell, B. (1979) Biochem. J. 184,469-472. 3. Minotti, G., and Aust, S. D. (1989) Chem. Biol. Interact. ‘71, l-19. 4. Gutteridge, J. M. C., and Wilkins, S. (1983) Biochim. Biophys. Acta 759,38-41. 5. Tien, M., Morehouse, L. A., Bucher, J. R., and Aust, S. D. (1982) Arch. Biochem. Biophys. 218,450-458. 6. Sarkar, B. (1970) Can. J. Biochem. 48, 1339-1350. I. Miller, D. M., and Aust, S. D. (1989) Arch. Biochem. Biophys. 271, 113-119. 8. Klein, S. M., Cohen, G., and Cederbaum, A. I. (1981) Biochemistry 20,6006-6012. 9. Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1984) J. Biol. Chem. 259, 3620-3624. 10. Baker, M. S., and Gebicki, J. M. (1986) Arch. Biochem. Biophys. 246,581-588. 11. Aruoma, 0. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M. (1989) J. Biol. Chem. 262,20509-20512. 12. Peterson, G. L. (1983) in Methods in Enzymology (Him, C. H. W., and Timasheff, S. N., Eds.), Vol. 91, pp, 95-119, Academic Press, New York. 13. Kirkpatrick, D. S., and Bishop, S. H. (1971) Anal. Chem. 43,17071709. 14. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. 15. Grisar, J. M., Petty, M. A., Bolkenius, F. N., Dow, J., Wagner, J., Wagner, E. R., Haegele, K. D., and De Jong, W. (1991) J. Med. Chem. 34,257-260. 16. Cederbaum, A. I., and Cohen, G. (1985) in CRC Handbook of Methods for Oxygen Radical Research (Greenwald, R. A., Ed.), pp. 8187, CRC Press, Boca Raton, FL. 17. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226,497-509. 18. Spiro, T. G., Pape, L., and Saltman, P. (1967) J. Am. Chem. Sot. 89,5555-5559. 19. Esterbauer, H., and Cheeseman, K. H. (1990) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 186, pp. 407421, Academic Press, San Diego. 20. Jorgensen, P. L. (1974) Biochim. Biophys. Actu 356,36-52. 21. Esmann, M. (1988) in Methods in Enzymology (Fleischer, S., and Fleiscber, B., Eds.), Vol. 156, pp. 105-115, Academic Press, San Diego. 22. Goldstein, D. B. (1968) Anal. Biochem. 24, 431-437.

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