Captopril and Enalaprilat Do Not Scavenge the Superoxide Anion Rakesh C. Kukreja, PhD, Hermes A. Kontos, MD, PhD, and Michael L. Hess, MD

The ability of captopril and enalaprilat, 2 angiotensin-converting enzyme (ACE) inhibitors, to scavenge superoxide anion radical was examined. With use of a number of superoxide-generating systems, such as xanthine-xanthine oxidase, phorbol myristate acetate-activated neutrophils, auto-oxidizing dihydroxyfumarate, and auto-oxidation of epinephrine to adrenochrome, captopril was seen not to scavenge superoxide directly, because it did not inhibit superoxide-dependent cytochrome c or nitroblue tetrazoiium reduction. Superoxide-dependent cytochrome c reduction was inhibited only when captopril was preincubated with a lower concentration of cytochrome c (22 NM). This effect was due to a decrease in the concentration of cytochrome c, because captopril reduced cytochrome c directly. When this effect was compensated for, no cytochrome c reduction induced by superoxide was observed. Captopril inhibited the auto-oxidation of ephtephrine to adrenochrome at pH 10.2 where this auto-oxidation is superoxide-dependent, and at pH 7.8 where it is superoxide-independent and superoxide dismutase insensitive. It appears that captopril, in this respect, acted as a nonspecific antioxidant, probably by reducing an intermediate in the complex oxidation of epinephrine to adrenochrome. Therefore, caution may be used in interpreting the role of captopril in the attenuation of reperfusioninduced myocardial dysfunction and in attributing this effect to the inhibition of free radical mechanism. (Am J Cardiol1990;65:24 I-27 I)

From the Division of Cardiology, Department of Medicine, Medical College of Virginia, Richmond, Virginia. This work was supported in part by Grants HL 40111 and HL 24917 from the National Institutes of Health, Bethesda, Maryland, and grant-in-aid 87061 from the American Heart Association, Dallas, Texas. Address for reprints: Michael L. Hess, MD, Box 281, Cardiology Division, Medical College of Virginia, Richmond, Virginia 23298.

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aptopril, an angiotensin-converting enzyme (ACE) inhibitor,’ has a number of effects independent of ACE inhibition; these include an anti-inflammatory actioq2v3 cardioprotective effect against reperfusion-induced arrhythmias,4 and attenuation of reperfusion-induced depression of myocardial function.5 Recently, Westlin and Mullane5 provided a possibleexplanation for theseactions.Theseinvestigators found that captopril diminished the reduction of cytochrome c induced by superoxide generated by xanthine oxidase plus xanthine or by activated neutrophils, and that it also decreasedthe rate of auto-oxidation of epinephrine. They assertedthat captopril scavengedsuperoxide and acted like superoxidedismutase(SOD). However, captopril contains a sulfhydryl group (SH) and, therefore, acts as a reducing agent. It is possible that becauseof this action, it may interfere with the cytochrome c reduction assay of superoxide. It is not clear from Westline and Mullane5 whether this effect of captopril was taken into consideration.A reexamination of the superoxidescavengingcapabilities of captopril, therefore, appeared in order. We also compared the superoxide scavengingcapabilities of another ACE inhibitor, enalaprilat, the deesterilied form of enalapril.

C

METHODS

We studied the ability of captopril and enalaprilat to scavengesuperoxidein a variety of superoxide-generating systemsdescribedin the ensuing paragraph. In someexperiments, the effects of dimethyl sulfoxide (DMSO [from Sigma]), a known hydroxyl radical scavenger,and of cysteine (Sigma), an SH-containing compoundthat is known not to scavengesuperoxidedirectly werecomparatively studied. Xanthine-xanthine oxidase: Superoxidegeneratedby xanthine oxidase action on xanthine was determined by monitoring cytochrome c reduction at 550 nm. A typical assayconsistedof 50 PM xanthine (sodium salt, Sigma), 0.005 p/ml xanthine oxidase (Boehringer Mannheim 20 p/ml), 0.05 mM cytochrome c (Sigma) in potassium phosphatebuffer (0.05 M, pH 7.8 at 22°C) containing 0.1 mM ethylenediaminotetra-acetate(EDTA). Captopril, enalaprilat or SOD were added in the reaction mixture before initiation of the reaction by xanthine oxidase. In some experiments, different concentrations of xanthine, cytochrome c or xanthine oxidase were used. Phorbol myristate acetate-stimulated human neutrophils: Heparinized human blood wasmixed with equal

volumes of 2% dextran in 0.9% saline solution and the erythrocytes were allowed to settle for 1.5 to 2 hours at room temperature. The neutrophil-rich plasma was cen-

Captoprlf_(

1 mu)

g 0.06 s 5 2 2004~ L? 9 DHF+SOD(lO@g/ml) 0 00

0

3

6

9

TIME (MINUTES) TIME

(MINUTES)

-ofcytociuomecbYcaPFIGURE 1. Dosetoprll. Captopdl initiated immedate reduction ot cytochrome otqeroxhkbyxanthine at!5!5omnwithouttbeganeratlon oxidass actlon on xantbine.

J c

FIGURE 3. Superoxide ganaratian trum autoeddizing dihy&w@anarate (DRF) and ettects ot captopril and swerotide dlsmutase(sDD).capt~l(soo~)bdnotckprarstheruP-e---at-O nm, whereas SOD (10 &ml) dii inhibit.

captopr11

0

3

TIME (MINUTES)

6 TIME (MINUTES)

9

12

15

FIGURE 4. Inability ot uptopril or enakqdiat to i&kit sweroxkkdwdWcytcduomecnductionlrom~myristateacetate-stlmuMsd naubophib.Supsroxidadismutass (SW) (10 pghni) but not captopd or c?mala@iat iWbRed superoxide-dapendcntcytochnnnec-.

pendedin Hank’s buffer containing glucose,calcium and magnesium. trifuged at 170 X g for 15 minutes. Contaminating erythA typical assayconsistedof 100PM cytochromec and rocytes were removed by hypotonic lysis and the neu- 0.5 X lo6 cells/ml in Hank’s balancedsalt solution. The trophils were washed with 0.9% saline solution before cells were activated by phorbol myristate acetate in layering onto Ficoll-Hypaque density gradient and cen- DMSO (200 rig/ml) and the rate of cytochrome c reductrifuged at 170 X g for 20 minutes. The pellet was resus- tion was monitored at 550 nm for 10 to 15 minutes.

control .osson 0 0

L

1

2

3 4 MINUTES

5

FIGURE 5. Left, pmincubation of cytodmm cwithcaph@landpartiali@DRionotitssupero~mluctron.The reacth mixtnue cantahd lower cytochrome c (22 fl), 0.03 U/ml xantldm oxldass and 152 pM xanthh. Astehk, ctor3Ominutes.Right,h&itityotcaphpkttoinhibitsupertocbromec rehcthatterpreina&Mionwith1mMcytochww oxide-dependent nitrob& tetrazohan reduction under similar comhons. SDD = sweroxide di.

ii

i

rate ot cy-

THE AMERICAN JOURNAL OF CARDIOLOGY MAY 22, 1990

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A SYMPDSIUM:

THE RENIN-ANGIGTENSIN SYSTEM -

o--o Control

TlSSUE SPECIFIC ISSUES

pH 10.2

FIGURE 6. ‘fop, thne -ofopinsphrtlleoxidatblatpH102andeRectot -, ruperoxkle cbmtasa (80th enalapdat, cystohw and dlmothyl dfox-

lde(DMSG).Capbprilat1GGandSoGpM cyst&s (100 plVl) &

conmntratlons, SOD (10 &ml)

inhlbited epinepkine oxi(1 mm) or DMSD were wlthsutanyoRoct.Theratoofapinephrim oxiclath was monitored at 320 nm. 606 tom, tins COWSSOfepinephrineOxidation atpH7.SandedlectsotSDD,captoprll and DMSD. SDD (10 &ml) and DMSD (100 fl) were wlthout any depredW effect on splnsphrlns oxldatioll at this pkt, whoroas captoprll continued to InNbit in a

dation. Enalapilat

0

2

4

6

10

8

TIME (MINUTES) pH 7.0

Control

~mannerfromlOOt0 1,000 pM. DL = racemic mixture of Dand L-isomers

of cystetne.

.05

.oo 0

2

0

Captopril, enalaprilat or SOD were added before activation of cells by phorbol myristate acetate. Auto-oxidation of dihydroxyiumarate: Superoxide generated during the auto-oxidation of dihydroxyfumarate was measured by nitroblue tetrazolium (NBT) reduction assay.*The standard assaymixture had a total volume of 3 ml and contained 50 PM NBT, 3.33 mM dihydroxyfumarate in 0.05M phosphatebuffer containing 0.1 mM EDTA (pH 7.8). The rate of NBT reduction wasmonitored spectrophotometricallyat 560 nm. Captopril or SOD were included in the reaction mixture to study their effects. Epinephrine auto-oxidation: The auto-oxidation of epinephrine to adrenochrome was quantitated in vitro according to the method of Misra and Fridovich’ and subsequentlymodified by Sun and Zigman. Briefly, 10 ~1of stock epinephrine ( 10e2M) was usedto initiate the reaction in buffer (0.1 M EDTA/SO mM sodium carbonate buffer, pH 10.2) in the presenceor absenceof captopril, enalaprilat or SOD. The rate of epinephrine oxidation was monitored at 320 nm up to 10 minutes. Epineph26 I

THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 65

10

rine oxidation monitored at this wavelengthwas found to be more sensitiveand gave more consistent results than that measuredat 480 nm. The effect of SOD and captopril on the oxidation of epinephrine at 2 different pHs (7.8 and 10.2) was studied in potassium phosphate,or sodium carbonate buffers (0.05 M) containing 0.1 M EDTA. The pH 7.8 buffer contained 8.8 X 10m5M ferrous sulfate as catalyst.* RESULTS

Captopril reduced cytochrome c directly in a dosedependentfashion (Fig. 1). Cysteinehad the sameeffect, whereasenalaprilat had no such effect, showing that the reduction of cytochromec induced by captopril wasprobably due to the presenceof the SH group. In contradiction to the resultsreported by Westlin and Mullane,5 we found that captopril had absolutely no superoxide-scavengingcapability. This was the casewhen superoxidewas generatedby xanthine plus xanthine oxidase(Fig. 2), by auto-oxidizing dihydroxyfumarate (Fig. 3), or by phorbol myristate acetate--activated neutro-

phils (Fig. 4). Becausecaptopril reduced cytochrome c directly (Fig. l), the resultant reduction in cytochrome c concentration is certain to result in a decreasedrate of reduction under specific assay conditions such as lower cytochrome c concentration, higher xanthine oxidaseand increasedpreincubation time with captopril. Fig. 5 (left) representssuch a partial inhibition of cytochrome c when captopril(1 mM) waspreincubatedfor 30 minutes before initiating reaction with xanthine oxidase.The diminution in the rate of reduction of cytochrome c, induced under thesecircumstances,was eliminated by adding additional cytochrome c to the reaction mixture. Under similar conditions, if NBT was used,which was not reduceddirectly by captopril, instead of cytochromec, no detectableeffect of captopril on the rate of reduction of this dye could be detected (Fig. 5, right). Clearly, the findings showedthat captopril did not scavengesuperoxide.The effect of cysteine was similar to that of captopril in all respects.When captopril was added to the reaction mixture immediately before the initiation of superoxidegeneration, no significant decreasein the rate of superoxide-inducedreduction of cytochrome c was observed (Fig. 2 and 5, left). Captopril and cysteine, but not enalaprilat, diminished the rate of auto-oxidation of epinephrine at pH 10.2;SOD had a similar inhibitory effect at this pH. The findings with captopril confirm the results of Westlin and Mullane.* Misra and Fridovich7 showed that the autooxidation of epinephrine is a complex process,which can proceedby at least 2 independent pathways. At high pH (10.2), the auto-oxidation of epinephrine to adrenochrome is superoxide-dependentand, therefore, inhibited by SOD (Fig. 6, top). However, at lower pH (7.8), the auto-oxidation of epinephrine to adrenochromeis metalcatalyzed, and is independentof superoxide.For this reasonwe testedthe effect of captopril on the auto-oxidation of epinephrine at the lower pH. Captopril, but not SOD or DMSO, inhibited the rate of oxidation of epinephrine (Fig. 6, bottom). DISCUSSION

Our findings clearly showthat captopril doesnot scavengesuperoxidedirectly. The earlier suggestionby Westlin and Mullane5 that captopril is a scavengerof superoxide is probably due to erroneous interpretation of the assaysfor superoxideused.Captopril, by virtue of its SH group, acts as a reducing agent and reducescytochrome c, thereby reducing the concentration of this indicator in the assayof superoxide. This effect is dependenton the concentrations of xanthine oxidase,cytochrome c, captopril and the duration of preincubation used in this assay

(Fig. 5, left). Westlin and Mullane5 did not take into account the effect of this interaction of captopril with cytochrome c and misinterpreted the resultant decrease in the rate of superoxide-induced reduction of cytochrome c asdue to scavengingof superoxideby captopril. Our findings show that at pH 10.2 captopril reduced the rate of auto-oxidation of epinephrine to adrenochrome, a finding similar to that of Westlin and Mullane.5 These investigators, however, interpreted this as indicating that captopril scavengessuperoxide. Further analysis of the effects of captopril on the auto-oxidation of epinephrine showsthat this interpretation is not warranted. Captopril interfered with the auto-oxidation of epinephrine at a lower pH where the auto-oxidation of epinephrineis independentof superoxideand whereSOD hasno effect.7A more reasonableinterpretation, which is consistent with all of the findings, is that captopril, by virtue of its SH group, acts as a nonspecific antioxidant. BecauseDMSO was ineffective in inhibiting the autooxidation of epinephrine at pH 7.8, hydroxyl radical is probably not involved. The effect of captopril is probably exerted via reduction of an intermediate product in the complex of auto-oxidation of epinephrine to adrenochrome. Whether this antioxidant effect of captopril is of clinical value cannot be evaluated from our experimentsand has to be tested by direct experimentation in various situations in which oxidant injury to tissuesoccurs.In this respect,however,at least with regard to the antioxidant effectsof captopril in the auto-oxidation of epinephrine,it is not superior to cysteine.

REFERENCES 1. Ondetti MA, Rubin B, Cushman DW. Design of specific inhibitors of angiotensin-converting enzyme: a new class of orally active antihypertensive agents. Science 1977;196:441-444. 2. Fantone JC, Schrier D, Weingarten B. Inhibition of vascular permeability changes in rats by captopril. J Clin Invest 1982,69:1207-1211. 3. Martin MFR. McKenna F, Bird HA, Surrall EK, Dixon JS, Wright V. Captopril: a new treatment for rheumatoid arthritis? Loncet 1984;1:1325m1327. 4. Woodward B, Zakaria M. Effect of some free radical scavengers on reperfusion-induced arrhythmias in the isolate rat heart. J MO/ Cell Car&o/ 1985; 17:485-493. 5. Westlin W, Mullane K. Does captopril attenuate reperfusion induced myocardial dysfunction by scavenging free radicals? Circulation 1988;77:supp/ I:I-130I-139. 6. Sun M, Zigman S. An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation. Anal Biochem 1978;90:81-89. 7. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Bid Chem 1972; 247:3170-3175. 8. Kukreja RC, Okabe E, Schrier GM, Hess ML. Oxygen radical mediated lipid peroxidation and inhibition of Ca 2+-ATPase activity of cardiac sarcoplasmic reticulum. Arch Biochem Biophys 1988:261:447-457.

THE AMERICAN JOURNAL OF CARDIOLOGY MAY 22, 1990

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Captopril and enalaprilat do not scavenge the superoxide anion.

The ability of captopril and enalaprilat, 2 angiotensin-converting enzyme (ACE) inhibitors, to scavenge superoxide anion radical was examined. With us...
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