Antiarrhythmic agents. Scavengers of hydroxyl radicals and inhibitors of NADPH-dependent lipid peroxidation in bovine lung microsomes.

Antiarrhythmic drugs, e.g. lidocaine, quinidine, and procainamide have been suggested as a means of reducing myocardial damage. The mode of action of these drugs have been attributed to their "membrane-stabilizing" properties. However, as tissue ischemia reperfusion is reported to generate toxic species of oxygen, we investigated the oxygen radical scavenging properties of these drugs and their effect on NADPH-dependent lipid peroxidation. These antiarrhythmic drugs are found to be ineffective as superoxide radical scavengers but are potent scavengers of hydroxyl radical with rate constants of 1.8 x 10(10) M-1 s-1, 1.61 x 10(10) M-1 s-1, and 1.45 x 10(10) M-1 s-1 for quinidine, lidocaine and procainamide, respectively, as determined by deoxyribose assay. In EPR study, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, lidocaine, quinidine, and procainamide caused a dose-dependent inhibition of DMPO-OH adduct formation. These drugs also caused a dose-dependent inhibition of NADPH-dependent lipid peroxidation when lung microsomes were incubated with NADPH in presence of Fe(3+)-ADP. We propose that the antiarrhythmic agents exert their beneficial effects, in part, by their ability to scavenge toxic species of oxygen and by reducing membrane lipid peroxidation.

possible mechanism of pulmonary injury (11)(12)(13). Since lipid peroxidation can be initiated by reactive species of oxygen which are produced during reperfusion of ischemic tissue, we developed the hypothesis that antiarrhythmic agents might be exerting their beneficial effects, in part, by a free radical scavenging mechanism thereby inhibiting lipid peroxidation. In this study we present evidence that antiarrhythmic drugs such as lidocaine, quinidine, and procainamide are potent inhibitors of hydroxyl radicals and are capable of inhibiting NADPH-dependent lipid peroxidation.

5,5-Dimethyl-l-pyrroline N-oxide (DMPO)' was obtained from
Sigma. The DMPO was purified by stirring aqueous solutions of DMPO (900 mM) with activated charcoal at 10 mg/ml and filtered through 0.22-p Millipore filter cartridges and then centrifuged at 2000 X g for 2 min. The purified DMPO did not give any EPR signal when scanned at 45 and 90 mM concentrations.
Preparation of Bovine Microsomes-One bovine lung lobe was collected on ice after sacrifice and was minced with scissors. The minced lung was homogenized for 3 min in a Waring blender with 5 volumes of ice-cold 0.15 M potassium phosphate buffer, pH 7.6, and filtered through a triple-layered cheese cloth. The filtrate was centrifuged at 24,000 X g for 10 min to remove mitochondria, nuclei, and cell debris. The supernatant was then centrifuged at 100,000 X g for 90 min, and the microsomal pellet was collected. The microsomes were washed by resuspending in 0.15 M phosphate buffer, pH 7.6, to the original volume and by sedimenting at 100,000 X g for 90 min. The washed microsomal pellet was then resuspended in Tris-HC1 buffer, pH 7.6, to yield a final concentration of 10 mg of microsomal protein/ml. The washing procedure removes most contaminants of hemoglobin, superoxide dismutase, and catalase from microsomal protein. Protein concentration was determined by Bradford protein assay using bovine serum albumin as the standard (17).
Assay of Lipid Peroxidation-Lipid peroxidation was determined by the thiobarbituric acid method (18,19). The reaction mixture, containing 3 mg of microsomal protein in Tris-HC1 buffer, pH 7.6, 200 FM NADPH, 1 mM ADP, 50 PM ferric chloride was incubated at 37 "C for 15 min. Lipid peroxidation was initiated by addition of NADPH and was terminated by addition of 2 ml of 0.5% (w/v) TBA and 2% trichloroacetic acid. This mixture was heated at 95 "C for 10 minutes. Three ml of chloroform was added after cooling and the mixture was vortexed for 30 s. Samples were centrifuged, and malondialdehyde (MDA) concentration was determined by reading at 535 nm against a blank that contained all reagents except NADPH and by using an extinction coefficient of 1.56 X lo6 M cm" (19). The The abbreviations used are: DMPO, 5,5-dimethyl-l-pyrroline Noxide; EPR, electron paramagnetic resonance; TBA, thiobarbituric acid; TEMP, 2,2,6,6-tetramethylpiperidine; O?, superoxide anion; 'OH, hydroxyl radical; lo2, singlet oxygen; MDA, malondialdehyde. effect of various concentrations of lidocaine, quinidine, and procainamide was tested in this system.
Assay of NADPH-cytochrome P-450 Reductase-NADPH-Cytochrome P-450 reductase was measured by its NADPH-cytochrome c reductase activity (20). One unit of NADPH-cytochrome c reductase is defined as 1 pmol of ferricytochrome c reduced per min. The reaction mixture contained the following reagents at final concentrations: potassium cyanide, 1 mM; NADPH, 4.2 X M; nicotinamide, 188 mM; ferricytochrome c, 6 X M. The effects of lidocaine, quinidine, and procainamide at various concentrations on the activity of cytochrome P-450 reductase was determined in this system. EPR Studies-Hydroxyl radicals were generated in a Fenton-type reaction and were detected as DMPO-OH adduct. The reaction mixture contained the following reagents at the final concentration: 31 p~ HzO,, 33.2 p M ferrous sulfate, 0.83 mM EDTA, 1.12 mM purified DMPO in 0.2 M boric acid/borax buffer, pH 7.8. The reaction was initiated by the addition of ferrous sulfate. Various levels of lidocaine, quinidine, and procainamide or other scavengers of hydroxyl radicals were used in the above system. The generation of hydroxyl radicals was observed as DMPO-OH adduct on a Bruker D-200 X-Band EPR spectrometer. Scan conditions, unless otherwise indicated, were as follows: microwave frequency, 100 KHz; power, 10 mW; modulation amplitude, 1.0 G; modulation frequency, 100 KHz; time constant, 0.64 s, scan time, 200 s; receiver gain, 4.0 X lo5; center field setting, 3483 G.
Photolysis studies were performed at room temperature, in the presence of dissolved air, in quartz capillary tubes. Samples were irradiated for various time periods at a distance of 30 cm from the lens of a Viewlex VR-25 remote-controlled slide projector. EPR measurements were made as described above.
Determination of Rate Constant-The degradation of deoxyribose by hydroxyl radicals and the production of a pink chromogen monitored spectrophotometrically has been used to determine the rate constants of various compounds (14)(15)(16) and are in agreement with the rate constants determined by the pulse radiolysis method. Hence, we used the deoxyribose method (14) to determine the rate constant for reaction of 'OH with the antiarrhythmic agents (lidocaine, quinidine, and procainamide).
Reaction mixtures contained, in a final volume of 1.0 ml, the following reagents at the final concentrations stated deoxyribose (2.8 mM), KH,PO,-KOH buffer, pH 7.4 (20 mM), FeC4 (25 pM), EDTA (30 pM), H202 (1 mM), and ascorbate (100 pM). Solutions of FeC13 and ascorbate were made up immediately before use in deaerated water. Reaction mixtures were incubated at 37 "C for 1 h, and one ml of 0.5% TBA (w/v) and 1 ml of 1.4% trichloroacetic acid (w/v) was added, and the mixtures were heated at 80 "C for 30 min. The rate of deoxyribose degradation was constant over the 1-h incubation period (14). A linear rate of MDA production was observed with incubation time and appeared to have reached a maximum after 10 m i n (Fig. 1C). Preincubation with lidocaine, quinidine, and procainamide inhibited the NADPH-induced lipid peroxidation in a dose-dependent manner (Fig. 2). Lidocaine was found to be the most potent inhibitor of NADPH-dependent lipid peroxidation followed by quinidine and procainamide. Quinine, a stereoisomer of quinidine, but a less effective antiarrythmic agent (21), was only 60-66% as effective as quinidine at 200, 300, and 400 p M concentrations in this reaction. The dose-dependent effects of lidocaine on the extent of lipid peroxidation was further examined. As shown in Fig. 2, lipid peroxidation was strongly inhibited by lidocaine and the FIG. 2. Effect of antiarrhythmic drugs (lidocaine, quinidine, and procainamide) on NADPH-dependent lipid peroxidation. The experimental conditions are as described under "Materials and Methods." The reaction mixture contained 3 mg/ml of microsomal proteins, 200 p M NADPH, 1 mM ADP, 50 p M FeC13, and the indicated concentrations of antiarrhythmnic drugs in Tris-HCI buffer, pH 7.6, and was incubated at 37 "C for 10 min. Lipid peroxidation was initiated by addition of NADPH and was terminated by the addition of 2 ml of 0.5% (w/v) TBA and 2 % trichloroacetic acid. This mixture was heated at 95 "C for 10 min. maximum inhibition actually achieved was 72%. When these data were presented on reciprocal coordinates (Fig. 2, inset), inhibitions in all cases appeared to be kinetically simple and the extrapolation of t h e data indicate that there was a maxim u m of 75% inhibition on lipid peroxidation by lidocaine, indicating the existance of a competing alternate pathway for lipid peroxidation.

Effect of Antiarrhythmic
Effect of Scavengers of Reactive Oxygen Species on MDA Production-It was shown previously that liver microsomes generate H202 in the presence of NADPH (22,23). Since 02: can dismutate to form Hz02 and in a metal-catalyzed reaction, H2OZ + 0, can form 'OH, it has been suggested that the reactive species of oxygen may be involved in lipid peroxidation initiation reaction.
In order to compare the action of Antiarrhythmic Drugs and Lipid Peroxidation antiarrhythmic drugs with other known scavengers, data were obtained on NADPH-dependent MDA production by lung microsomes in the presence or absence of several free radical scavengers. Thus, as shown in Table I, the hydroxyl radical scavengers thiourea and ethanol inhibited MDA formation 100 and 61%, respectively, at 10 mM concentrations. DMPO, a spin trap which removes 0; and 'OH by forming DMPO-OOH and DMPO-OH adduct, respectively, also inhibited the formation of MDA by 100%. Catalase at 5000 units/ml inhibited the formation of MDA by 81%. Superoxide dismutase 1-10 pg/ml did not inhibit the formation of MDA, rather a slight increase was noted. This is in agreement with our previous report (24).
Mannitol and benzoate increased the formation of MDA by 20 and 30%, respectively, at 10 mM. This is in agreement with Cohen and Cederbaum (25) who observed similar effects of mannitol in a lipid peroxidation-dependent chemiluminescence reaction. It is likely that mannitol and benzoate, being hydrophilic, cannot partition to the membrane bilayer for the effective scavenging of 'OH at the site of its production. In order to test this hypothesis, 1 mM mannitol was added to a n-octanol/water (50:50) mixture, vigorously shaken for 5 min, and allowed the phases to separate. Mannitol was measured in the two phases colorimetrically by the chromotropic acidformaldehyde reaction using periodic acid to oxidize the sugar alcohol (26). This assay was found to be sensitive at 20 p~ mannitol, and a linear standard curve was obtained with mannitol concentrations from 20 p M to 1 mM. Almost 100% of the mannitol was found to be partitioned to the aquous phase and no trace of mannitol was detected in the n-octanol phase. Since n-octanol has a dielectric constant similar to most biomembranes, it is likely that the lack of protection of microsomes against lipid peroxidation by mannitol is due to its inability to reach the membrane lipid bilayer.
Effect of Antiarrhythmic Agents on the Activity of NADPHcytochrome P-450 Reductase-In NADPH-dependent lipid peroxidation, NADPH-cytochrome P-450 reductase is the key enzyme causing one electron reduction of molecular oxygen or Fe3+ (27). This enzyme serves as electron carrier from NADPH to oxygen via cytochrome P-450. Although the antiarrhythmic drugs inhibited the NADPH-dependent microsomal lipid peroxidation in a dose-dependent manner, there is still reason to believe that these drugs could inhibit the NADPH-cytochrome P-450 reductase and thus lower the flux of univalent oxygen reduction to generate fewer oxyradicals. To exclude the possibility of this subtle artifact we have examined the effect of antiarrhythmic agents on the activity of NADPH-cytochrome P-450 reductase. As shown in Table   TABLE I  11, the activity of the enzymes remain unchanged when various concentrations of lidocaine, quinidine, and procainamide were incubated with microsomes in presence of NADPH. These studies established that the inhibition of microsomal lipid peroxidation by antiarrhythmic drugs was not due to decreased production of active oxygen species and is more likely due to scavenging of these species. Effects of Antiarrhythmic Agents on Active Oxygen Species-In an attempt to identify the radical species directly involved in initiating lipid peroxidation, and which can be scavenged by antiarrhythmic agents, we investigated the role of these agents in various known activated oxygen-generating systems.
Superoxide anions are known to be produced when xanthine oxidase acts on xanthine in the presence of molecular oxygen. The 0; so generated can reduce ferricytochrome c, and this has been used as a convenient assay for superoxide dismutase (28). We tested the effects of antiarrhythmic agents lidocaine, quinidine, and procainamide at 0.5 and 1.0 mM concentrations and found that these agents are not effective in scavenging 0; (data not shown).
The hydroxyl radicals generated in a Fenton-type system (Fez+ +H202 + 'OH + OH-+ Fe3+) yield spin adducts with DMPO (22). Thus, as presented in Fig. 3  Addition of ' OH scavengers inhibited the signal intensity in a dose-dependent manner. Thus, 1 mM thiourea and 3 mM mannitol inhibited the signal almost completely (data not shown). The effect of the antiarrhythmic agents lidocaine, quinidine, and procainamide were tested in this system. None of the antiarrhythmic agents reacted with H202 which is in accord with the previous findings (1). As shown in Fig. 3A-C, lidocaine, quinidine, and procainamide inhibited the DMPO-OH adduct formation in a dose-dependent manner. The percent inhibition was calculated from the signal heights and is presented in Fig. 30. The molar concentration of drugs required to cause 50% inhibition was found to be 108, 108, and 300 p~ for lidocaine, quinidine, and procainamide, respectively.
If inhibition by antiarrhythmic agents of 'OH, as shown in Fig. 3, are truly a reflection of interaction of these agents with the 'OH, then identical results should be obtained with a different assay. That this was the case is illustrated in Fig. 4 where a different assay, a deoxyribose colorimetric assay (14), was adopted. In this assay a mixture of FeCh-EDTA, H202, and ascorbic acid at pH 7.4, 'OH radicals which can be detected by their ability to degrade the sugar deoxyribose into  fragments and which generate a pink chromogen upon heating with TBA at low pH.
Fe3+-EDTA + ascorbate + Fez+-EDTA + oxidized ascorbate (1) Fez*-EDTA + H202 + Fe3+-EDTA + 'OH + OH (2) 'OH + deoxyribose + degraded sugar + color product (3) The 'OH so generated (Reaction 2) is equally accessible to FIG. 4. Hydroxyl radical scavenging by antiarrhythmic drugs: determination of rate constants. Deoxyribose degradation in the presence of various concentrations of antiarrhythmic drugs was followed as described under "Materials and Methods" using a final deoxyribose concentration of 2.8 mM in the reaction mixture. The rate constant was determined form the slope of the line ( k = slope X k D R X [DR] X A ) as described in the text giving the value of lidocaine, 1.6 X 10" M" s-' ; quinidine, 1.8 X 10" M" s-'; procainamide, 1.45 X 10" M-' s" in this experiment. deoxyribose (the detector molecule) and to any other scavenger of 'OH added. Thus, the ability of a substance to inhibit competitively with deoxyribose under these conditions is a measure of its ability to scavenge 'OH and can be used to calculate the rate constant for reaction of 'OH with scavengers (14). Lidocaine, quinidine, and procainamide were able to compete with deoxyribose effectively in preventing the TBA-reactive color product formation. The second order rate constants for the reaction of these agents with 'OH were calculated (14) and were found to be 1.8 x 10" M" s-', 1.61 X 10" M" s-', and 1.45 X lo1' M" s" for quinidine, lidocaine, and procainamide, respectively (Fig. 4). Control experiments showed that none of the antiarrhythmic drugs interfere with the measurement of deoxyribose degradation or itself react with 'OH to give TBA-reactive color products. Thus, when 500 PM each of the drug was added to the reaction mixture at the end of the incubation time, before the addition of TBA, little protection of deoxyribose degradation was observed. Moreover, when these drugs were allowed to react with the 'OH-generating system in the absence of deoxyribose, no TBA-reactive products were observed a t 535 nm.
Since singlet oxygen is thought to be involved in the lipid peroxidation process, we have investigated the role of antiarrhythmic agents in known singlet oxygen-generating systems. The generation of singlet oxygen by photochemical reactions of rose bengal or methylene blue was studied by EPR spectroscopy using TEMP as a singlet oxygen trap. We have demonstrated the formation of TEMPO as a nitroxyl radical by the attack of singlet molecular oxygen, generated during photoactivation of various sensitizers, on TEMP (29,30). The characteristic EPR spectral pattern of three lines of equal intensity for the TEMPO nitroxide radical was observed when an air-saturated aqueous solution of rose bengal was irradiated in the presence of TEMP at room temperature (Fig. 5, inset). The hyperfine splitting constants and g-value of the radical were found to be AN = 17.2 G and g = 2.0056, respectively, consistent with our previously reported values (29,30). Lidocaine, quinidine, and procainamide inhibited the TEMPO formation in a dose-dependent manner (Fig. 5). However, as presented in Table 111, when rose bengal was replaced with methylene blue (600 FM) as a photosensitizer and TEMPO accumulation was monitored for a period of 10 min, quinidine (1-10 mM) and procainamide (1-10 mM) did not inhibit the formation of TEMPO. This difference is suggestive of sensitizer quenching rather than singlet quenching. Lidocaine on

TABLE 111
Effects of antiarrhythmic drugs on methylene blue sensitized singlet oxygen generating system Experimental conditions are as described under "Materials and Methods." Lidocaine, quinidine, and procainamide at indicated concentrations were added to 600 p~ methylene blue, 65 mM TEMP and 0.05 M potassium phosphate buffer, pH 7.8, with M EDTA. The TEMPO formation was recorded after irradiating the mixture for 5 min. The percent inhibition was calculated from the intensity of the first signal. the other hand was found to inhibit TEMPO formation a t higher concentrations in a dose-dependent manner. Thus, a t a concentration of 4.18 mM, lidocaine inhibited 50% of the TEMPO signal.

DISCUSSION
In the present study we have demonstrated that antiarrhythmic agents (lidocaine, quinidine, and procainamide) inhibit NADPH-dependent lipid peroxidation at micromolar concentrations. Among the three antiarrhythmic agents tested, the order of ability to inhibit lipid peroxidation was: lidocaine > quinidine > procainamide (Fig. 2), which is consistent with the therapeutic potential of the drugs (31-33).
Lidocaine, quinidine, and procainamide are found to be scavengers of hydroxyl radical. This was demonstrated both in the EPR spin-trapping study as well as in the deoxyribose degradation assay (Figs. 3 and 4). Thus, when 'OH radicals were generated in a Fenton-type reaction and detected as DMPO-OH adduct by EPR spectroscopic techniques, lidocaine, quinidine, and procainamide inhibited these reactions in a dose-dependent mannner. Compared with some of the known hydroxyl radical scavengers, lidocaine, quinidine, and procainamide were more sensitive than ethanol or thiourea.
Lidocaine, quinidine, and procainamide inhibited '02-dependent TEMPO formation (Fig. 5) when rose bengal was used as a sensitizer. However, when rose bengal was replaced by methylene blue, there was no inhibition of TEMPO for-mation by both quinidine (1-10 mM) and procainamide (1-10 mM). Lidocaine inhibited this reaction at high concentrations, which is consistent with our previous findings (34). The inhibition of TEMPO formation may be due to ionic interaction between the cationic drugs quinidine and procainamide with rose bengal ( 2 7 , since these drugs had minimal effects when cationic sensitizer methylene blue (1+) was used to generate lo2. Therefore it appears likely that quinidine and procainamide are quenching the sensitizer rather than the singlet oxygen. Lidocaine, however, was able to inhibit TEMPO formation in both rose bengal and methylene blue systems in a dose-dependent, manner indicating that it may posses singlet oxygen scavenging/quenching activity. Although millimolar concentrations of the drug are required to inhibit singlet oxygen-dependent TEMPO formation, these concentrations are comparable with the concentrations of other known singlet quenchers such as histidine, dimethylfuran, and diphenylfuran (24) in inhibiting lipid peroxidation. At pharmacologically relevant concentrations, however, it may be that none of the three antiarrhymic drugs are efficient in scavenging singlet oxygen.
The cytoprotective effects of lidocaine, quinidine, and procainamide in myocardial ischemia have been attributed to membrane stabilizing properties (31-33). However, the mechanism of such "membrane stabilization" has not been elucidated. Recently, Woodward and Zakaria (35) and Manning et al. (36) have proposed the hypothesis that oxygen-derived free radicals generated during the early moments of reperfusion may initiate membrane injury, leading to the development of severe ventricular arrhythmias. During myocardial ischemia, especially after reoxygenation of the heart, there is an apparent accumulation of lipid peroxides in the tissue (2, 7, 9, 10). Canine sarcolemmal membranes were also found to be readily peroxidized by reactive species of oxygen, and the phospholipid-rich sarcolemma of ventricular myocytes were proposed to be a major site of free radical attack (37). Hydroxyl radicals are proposed to be the direct initiator of lipid peroxidation by concerted addition-abstraction reactions with the diene bonds of unsaturated lipids (38-41). It was suggested that singlet oxygen and 'OH are involved in the propagation of lipid peroxidation (38), which in turn can produce membrane damage and cell dysfunctions (39). The results of the present study show that lidocaine, quinidine, and procainamide are powerful antioxidants, which can scavenge hydroxyl radical and prevent membrane lipid peroxidation. Therefore, the membrane stabilization ability of lidocaine as reported earlier may, in part, be attributed to the reactive oxygen scavenging properties of this compound.
Recent studies have indicated the involvement of reactive oxygen species in myocardial ischemia. Thus, Bernier et al. (42) found that superoxide dismutase, catalase, mannitol, methionine, glutathione, and desferroxamine reduced the incidence of reperfusion-induced ventricular fibrillation from 80% to 0, 7, 7, 7, 20, and 7%, respectively. They also demonstrated a clear dose dependency for each intervention. In another study (10) it was found that superoxide dismutase plus catalase enhanced the efficacy of hypothermic cardioplegia in protecting the globally ischemic reperfused heart. The production of oxygen-centered free radicals during ischemia and reperfusion of myocardium have been demonstrated, and a burst of oxygen radical generation was shown to occur within moments of reperfusion (41). Electron spin resonance studies have conformed the production of oxygen free radicals in ischemic/reperfusedrat hearts (4,5,41). These studies suggest that excessive free radicals are produced in the extracellular compartment, and these radicals in turn are responsible for the sarcolemmal membrane damage.
In our present study, lidocaine, quinidine, and procainamide inhibited NADPH-dependent lipid peroxidation. The scavengers of hydroxyl radicals, such as ethanol, thiourea, and DMPO (43), were found to inhibit MDA formation. However, mannitol and benzoate were found to increase the MDA formation when used in higher concentration (Table I). Mannitol is known to increase the production of MDA when citrate-Fez+ is used as a source of chelated iron, but the mechanism of this increase is not known (44). Our data indicate that mannitol cannot partition to n-octanol phase. Since n-octanol has a dielectric constant similar to most biomembranes, it is possible that the inability of mannitol to protect microsomal membrane against lipid peroxidation may, in part, be due to its inability to reach the site of free radical production. Isolated rat liver microsomes in the presence of NADPH and iron can catalyze the production of 'OH-like species by a reaction sensitive to catalase but not to superoxide dismutase (24). The involvement of hydroxyl radical in the NADPH-dependent lipid peroxidation has been a subject of controversy. Our observed inhibition of lipid peroxidation by the scavengers of hydroxyl radical are in accord with the findings of some investigators (25,45-47), but not with those of others (48,49). The controversy regarding the involvement of hydroxyl radicals in lipid peroxidation may, in part, be due to the different lipid composition of different membranes and the multiplicity of mechanisms of oxygen activation which cause lipid peroxidation. The composition of the reaction medium (presence and absence of exogenous iron) may also play an important role in generating these reactive species. The mechanism proposed below seems to be operating in our system in generating hydroxyl radicals. The NADPH-microsomal system generates 0; which can dismutate as in Reaction 1 or it can react with H202 as in Reaction 4. The later reaction generates the powerful oxidant 'OH which can continue the chain reaction to peroxidize the membrane lipids. Reactions 2 and 3 presented here are similar to Fenton's reaction (50-51). Since superoxide dismutase slightly enhanced the lipid peroxidation and catalase inhibited the reaction, we propose that reduced cytochrome P450 would form a complex, superoxoferricheme (Fe2+-02 Fe3+-Oz) by reacting wih molecular oxygen and that a small part of this complex will dissociate to form Fe3+ and 0 5 (52). 0; so generated can form H2OZ catalyzed by superoxide dismutase (Reaction 1). Here superoxide dismutase should enhance lipid peroxidation by forming H202, and catalase would inhibit the reaction by preventing the formation of 'OH (Reaction 3). Peroxidation of membrane lipids by 'OH then would be initiated by hydrogen abstraction from a methylene carbon cation. That the hydroxyl radical scavengers such as thiourea, ethanol, and DMPO inhibited lipid peroxidation is strong evidence that the reactive species, regardless of how it is generated, must be 'OH.
The fact that lidocaine, quinidine, and procainamide inhibited the lipid peroxidation as well as scavenged the 'OH, generated in a chemical reaction, suggests that these drugs protect the membrane lipids by scavenging 'OH. However, it does not conclusively establish a cause-effect between these two phenomena. Two points need to be considered; 1) the concentration dependence for inhibition of lipid peroxidation by these drugs is not paralleled by a similar concentration dependence for scavanging hydroxyl radical, and 2) it is possible that the inhibition of lipid peroxidation could be due to the ability of these drugs to interact directly with lipids thus indirectly protecting the membrane from 'OH attack. The first phenomenon may be explained by the fact that lidocaine is known to be further metabolized in the body rapidly and extensively into compounds (monoethylglycylxylidide and glycylxylidide) which have antiarrythmic activities (53); it is possible that in our microsomal-NADPH system these metabolic products are produced and are better antioxidants than lidocaine itself. Further investigation is needed to resolve this issue. To lessen the possibility of the second issue, a rather subtle artifact, we have added stearic acid to the lipid peroxidation reaction mixture to examine if addition of a saturated lipid diminishes the effectiveness of these drugs. In a similar reaction system the activity of these drugs (500 p~) remained unchanged in the presence of 500 p~ stearic acid (data not presented). These results strongly suggest that the membrane stabilization effects of these drugs may, in part, be due to their ability to remove the reactive oxygen species.