oxygen free radicals and myocardial damage: protective role of thiol-containing agents

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Oxygen Free Radicals and Myocardial Damage: Protective Role of Thiol-Containing Agents ROBERTO FERRARI, M.D., Ph.D., C. CECONi, M.D., S. CURELLO, M.D., A. CARGNONI, Ph.D., O. ALFIERI, M.D., A. PARDINI, M.D., P. MARZOLLO, M.D., O. VISIOLI, M.D., Brescia, Italy

It has been suggested that the sudden presence of oxygen during reperfusion after a period of ischemia may be toxic for the myocardial cell.

The oxygen molecule is capable of producing reactions in the cell, forming highly reactive free radicals, and inducing lipid peroxidation of membranes, altering their integrity and increasing their fluidity and permeability.

The ischemic and reperfused cardiac cell is the prime candidate for this reaction sequence and may explain the molecular mechanism underlying the pathologic events related to membrane dysfunction and calcium homeosta- sis.

However, the myocardium has a series of defense mechanisms including the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase plus other endogenous antioxidants such as vitamin E, ascorbic acid, and cysteine to protect the cell against the cy- toxic oxygen metabolites.

The prerequisite for oxygen free radical in- volvement in ischemia and reperfusion damage is that ischemia alters the defense mechanisms against oxygen toxicity. It is known that ischemia may impair mitochondrial SOD and, with reperfusion, oxidative stress may occur as shown by tissue accumulation and release of oxidized glutathione. This tripeptide molecule is the cofactor of glutathione peroxidase, the enzyme that removes hydrogen and lipid perox- ides. Its formation and subsequent release is a reliable index of oxidative damage.

In our study, we investigated the effects of N-acetylcysteine on oxidative damage in the isolated rabbit heart. N-acetylcysteine increases, in a dose-dependent manner (from 10 -~ to 10 -5 M), the myocardial glutathione content and provides an important degree of

From the Cattedra di Cardiologia, Universitfi degli Studi di Brescia; II Divisione di Cardiochirurgia, Ospedali Civili di Brescia; and Divisione di Anestesia e Rianimazione Cardiochirurgica, Ospedali CiviIi di Brescia, Brescia, Italy.

Requests for reprints should be addressed to Roberto Ferrari, M.D., Cattedra di Cardiologia, Universita degli Studi di Brescia, Ospedali Civili, P.le Ospedali Civili, I, 25100 Brescia, Italy.

protection against ischemia and reperfusion. Oxidative stress does not occur, mitochondrial function is maintained, enzyme release is reduced, and contractile recovery is increased. Similarly, we administered N-acetylcysteine in the pulmonary artery of coronary artery disease patients undergoing coronary bypass grafting (150 mg/kg in 1 hour followed by 150 mg/kg in 4 hours). The degree of oxidative stress on reperfusion was reduced and recovery of cardiac function improved.

In this article, we review the cardioprotective role of thiol-containing agents.

E vidence shows that oxygen free radicals are important mediators of several forms of tissue

damage, such as associated with inflammatory responses, ischemic injuries to different organs and tissues, and injuries resulting from intracellular metabolism of chemical and physical agents, such as drugs and radiation.

There has been added interest in the concept that oxygen free radicals play a role in the pathogenesis of myocardial ischemia and infarction.

Studies have reported reperfusion models, since oxygen is reintroduced into the system with coronary reperfusion following myocardial ischemia. Subsequently, the concept of an oxygen free radical-mediated cardiotoxicity has important clinical implications in myocardial ischemia followed by reperfusion.

Interventions such as streptokinase, tissue plasminogen activator, and percutaneous transluminal angioplasty are used to re-establish coronary flow in patients with myocardial infarction. Addition- ally, ischemia and reperfusion sequences occur in patients with vasospastic angina or during coronary angioplasty or cardiopulmonary bypass.

MYOCARDIAL SOURCES OF OXYGEN FREE RADICALS UNDER NORMAL CONDITIONS

Even in the normal aerobic myocardium, there is continuous production of oxygen free radicals. Su- peroxide anions (0~- • ) are produced in the electron transport systems of the mitochondria. Alterna- tively, they can be formed as by-products of various

September 30, 1991 The American Journat of Medicine Volume 91 (suppl 3C) 3C-95S

SYMPOSIUM ON OXIDANTS AND ANTIOXIDANTS / FERRARI ET AL

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enzyme-substrate reactions (xanthine oxidase) and by auto-oxidations of a variety of low-molecular- weight molecules. Hydrogen peroxide (H202) may be produced as a direct product or as an enzyme- catalyzed superoxide dismutase (SOD) dismutation.

The mitochondrial electron transport system is the most important site of free radical production under physiologic conditions. About 2% of the oxygen (02) utilized by intact, aerobic mitochondria is partially reduced by electrons that escape from electron carriers in the respiratory chain [1]. The primary Production site of 02 by cardiac mitochondria, responsible for 75% of 02 generation, is the region between quinones and cytochrome b, on the internal mitochondrial membrane. O~ • are formed by auto-oxidation of semiquinones rather than as a direct catalytic product [2]. Additionally, other auto-oxidizable electron carriers exist in the inner mitochondrial membrane, such as reduced nicotina-

mide-adenine dinucleotide (NADH) dehydrogenase, that are responsible for the remainder of the radical generation [3].

Since mitochondria are rich in SOD, the majority of 0~-. generated as a consequence of mitochondrial electron transport is enzymatically dis- mutated to H202 and 02. Hydroxyl radicals (- OH) are formed within the mitochondria arising from 0~. and H202 reaction.

Electron transport mechanisms that operate within the endoplasmic reticulum and within the nuclear membranes of eukaryotic cells also generate 0~-- and H202. Peroxisomes contain oxidases that generate H202 directly, without O~ • interme- diate formation [4]. Up co 40% of the H202 generated in peroxisomes diffuses in the cytoplasm and injures the cytosolic components.

O~ • in the myocardium is also the final product of the reaction between xanthine and xanthine oxidase (XO), an enzyme localized in the vascular en-

3C-96S September 30, i991 The American Journal of Medicine Volume 91 (suppl 3C)


Figure 2. Scheme of glutathione reduction oxidative cycle. G-6-P: glucose-6-phosphate; Prot- S-SG: protein-sulfhydryl-glutathione; Prot-SH: protein-SH; other abbreviations as in Figure I.

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dothelium [5,6]. It should be specified that the ha- tive form of the enzyme is a dehydrogenase that cannot reduce 02 and cannot produce O2-.. It ap- pears that healthy tissues contain approximately 10% of their total enzyme in the oxidase form [7]. Studies on the distribution of XO among various tissues and species show evidence that enzyme activity varies widely and certain organs in some species show no activity. Rabbit, pig, and human heart have little, if any, XO activity [7,8].

MYOCARDIAL ANTIOXIDANT MECHANISM The aerobic myocardium controls and survives

the continuous oxygen free radical production due to a delicate balance between cellular systems gen- erating the various oxidants and those maintaining the antioxidant defense mechanism (Figure 1).

In the heart, these defense mechanisms include the enzymes SOD, catalase, and glutathione peroxidase (GPD) plus other endogenous antioxidants such as vitamin E, ascorbic acid, and cysteine [7,9].

The primary mechanism for 0~" clearance is SOD, which catalyzes the dismutation of 0~" to H202 and 02 [10]. The reaction proceeds spontane- ously, but SOD increases the rate of intracellular dismutation by a factor of 109. At least three forms of SOD have been characterized. One contains copper and zinc and it is present in the cytos01. An- other contains manganese and it is present in the mitochondria. The third contains iron and it is associated with the cytoplasma of Escherichia coli.

Two enzyme systems are important in the metabolism of H202 produced by the univalent reduction of 0~- • [7,11]. The first is catalase present in cytosol that catalyzes the reduction of H202 to water. Catalase is present at only very low concentrations in the myocardium and GPD (a selenium-dependent enzyme) at a significant concentration in the cytosol of the heart [12].

The hexose monophosphate shunt produces, through glucose-6-phosphate oxidation, the reduc-

ing equivalents as nicotinamide adeninine dinucleotide phosphate (NADPH)to activate glutathione reductase. Reduced glutathione (GSH) is utilized by GSH peroxidase to form oxidized glutathione (GSSG). Changes in glutalhione status provide important information off the cellular oxidative events, and tissue accumulation and/or release of GSSG in the coronary effiuent is a sensitive and accurate index of oxidative stress [13-18]. GSH is in dynamic equilibrium with all cellular sulfhydryl groups. Glutathione and mixed disulfides with pro- teins constitute an important part of the total cellular GSH pool and the equilibrium is regulated by thiol transferases.

As the determinant of the sulfhydryl/disulfide ratio [19], GSH modulates the activity of a number

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of enzymes and is also involved in the transport of amino acids across the cell membrane [13]. GSH as a cosubstrate of GPD is protective against oxygen free radicals and prevents peroxidation of membrane lipids. SOD activity in the heart is nearly four times less than in the liver, whereas catalase activity is extremely low [19] (Figure 2).

Vitamine E, an antioxidant that has long been known in biologic systems, has been identified at significant concentrations in the myocardial, cyto- s01icl and mitochondrial membranes [20-22]. In vitro studies have shown that vitamin E functions

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as a free radical scavenger and protects the heart membrane from lipid peroxidation by free radicals [22-24]. It functions synergistically with ascorbic acid (vitamin C), which reacts with vitamin E radicals to regenerate vitamin E. Vitamin C radicals are reduced by NADH r~ductase [25]. Due to its lipophilic nature, vitamin E serves as an antioxidant within membranes and vitamin C serves as a water-soluble electron-transport system in the cytosol or in the extracellular fluid.

Although there is sufficient in vitro and in vivo evidence to support vitamin E as an important antioxidant, a protective role of this compound, at

September 30, I991 The American Journal of Medicine Volume 91 (suppi 3C) 3C-97S

SYMPOSIUM ON OXIDANTS AND ANTIOX!DANTS/FERRARI ET AL

physiologic concentration in humans, has not been well documented.

POTENTIAL SOURCES OF OXYGEN RADICALS IN ISCHEMIC AND REPERFUSED MYOCARDIUM

Myocardial ischemia arises when oxygen delivery to the myocardium is insufficient to meet the mitochondrial oxidation [26]. During ischemia, there is a lack of oxygen availability, but oxygen free radicals are still formed from the residual molecular oxygen.

There are many potential sources of free radicals in the myocardium (Figure 3), making it difficult to determine the most important site of production of these radicals. The problem is more complex be- cause oxygen-derived free radicals may be produced by different sources after different periods of ischemia and reperfusion. Thus, upon reperfusion, after a period of ischemia, only one or two sources may be involved, but if reperfusion is further delayed, other sources could become more important.

Radicals could be generated from the mitochondria, myocardial cell membrane, endothelial cells, and white cells.

During ischemia, the components of the mitochondrial electron-transport chain are reduced [27,28], increasing electron leakage from the respiratory chain, which reacts with residual molecular oxygen and forms 0 ~ ' , Reperfusion re-energizes the mitochondria , but electron egress through cytochrome oxidase is reduced due to lack of adenosine diphosphate (ADP), forming oxygen free radicals. In the early phases of ischemia, the increased oxygen free radical production from the mitochondria is neutralized by SOD. Increasing the duration of ischemia progresses the decline of SOD activity, leaving the mitochondria less equipped to deal with the increased radical flux [22,29,30]. Evidence shows increased production of reduced oxygen in- termediates from the heart mitochondria harvested after ischemia and reperfusion [31-35]. Production of oxygen free radicals from the mitochondria remains unquestioned.

In the capillary endothelial cell, the enzyme xanthine dehydrogenase (XD) is converted during ischemia to XO, which catalyzes the conversion of hypoxanthine and Xanthine to uric acid, using oxygen as electron acceptor. On reperfusion, the delivered oxygen is reduced by this system, producing oxygen free radicals [36]. There is evidence that allopurinol, an inhibitor of XO, protects the myocardium against reperfusion damage [37-47]. A]lo- purinol could be protective by mechanisms other than inhibition of the enzyme [44,46,47]. Not all studies with this compound are positive [48-51].

The distribution of XO varies widely, with the rabbit, pig, and human myocardium having essen- tially no activity. Yet, these species are not immune to reperfusion injury. Conversion into XD in the hearts is low, making it unlikely that XO is an ira- portant source of free radicals during early reperfusion, when damage occurs.

Neutrophils, when activated, generate several types of oxygen free radicals, being defenses against bacterial infection and inflammatory reactions, such as acute myocardial infarction [28]. These recruited neutrophils to the infarct site may damage the myocardium, producing oxygen free radicals. Removal of white cells from the blood reduces infarct size [51-54], but neutrophils are ab- sent from many preparations in which oxidative damage during reperfusion has been demonstrated. It is uncertain whether neutrophils are activated at the time of reperfusion or later, when damage has already occurred [55].

Free radicals may be generated within the mere- branes, in association with the arachidonic acid cas- cade and with the auto-oxidation of catecholamines. During ischemia, activation of phospholipases increases the release of arachidonate, and there is an increased release of norepinephrine [56-60].

Auto-oxidation of catecholamines, which are abundantly released from the ischemic myocardium, could provide oxygen free radicals through adrenochrome formation [61,62]. Vitamin E protects against isoprenaline-induced myocardial damage, while its depletion exacerbates the damage [63]. As with arachidonic acid metabolism, the role of catecholamines in oxygen free radical production remains unknown.

EFFECTS OF MYOCARDIAL ISCHEMIA AND REPERFUSION ON THE DEFENSE MECHANISM AGAINST OXYGEN TOXICITY AND OXIDATIVE STRESS

To investigate the role of oxygen in reperfusion injury in isolated and perfused rabbit hearts [14,15,30], we have determined the effects of ischemia on the activity of mitochondrial and cytosolic SOD, and of ~GPD and glutathione reductase (GRD), the two major defenses against oxygen free radical production. In addition, we have measured the tissue GSH/GSSG ratio as an index of oxidative stress.

Reduction of coronary flow to 1 mL/min induced a rapid decline in developed pressure, with contractile activity completely ceasing 9 minutes after the onset of ischemia. Resting pressure rose pro- gressively 20 minutes after the onset of ischemia. Ninety minutes of ischemia specifically reduced the activity of mitochondrial Mn-SOD, while the same

3C-985 September 30, 1991 The American Journal of Medicine Volume 91 (suppl 3C)

SYMPOSIUM ON OXIDANTS AND ANTiOXIDANTS/FERRARI ET AI.

ADRENAUNE ADRENOCROME

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SYMPOSIUM ON OXIDANTS AND ANTIOXIDANTS/FERRARI ET AL

period of ischemia did not affect the activity of the cytosolic Cu-Zn-SOD, or of GPD or GRD. Ische- mia induced a reduction in myocardial GSH/GSSG ratio. This was due to a significant reduction in tissue content of GSH, with GSSG being unchanged.

Reperfusion induced a significant increase in diastolic pressure with no recovery of development pressure. On reperfusion, tissue GSSG significantly increased, resulting in a further decline in the GSH/ GSSG ratio. Reinstitution of coronary flow did not significantly modify the SOD, GRD, and GPD ac- tivities. The changes in mitochondrial SOD and GSH/GSSG ratio during reperfusion were coinci- dent with important changes in the rate of release of GSH and GSSG. Ischemia did not significantly change the rate of GSH or GSSG release, but following reperfusion there was a marked and sustained release of GSH and GSSG from the heart.

These results suggest that ischemia induces met- abolic changes capable of reducing the defense mechanisms against oxygen toxicity. The prime change lies at the level of mitochondrial SOD, its activity being reduced by 50% after Severe ischemia.

Under these conditions, the readmission of 02 stimulates the production of oxygen radicals above the neutralizing capacity of mitochondrial SOD. Consequently, the second line of defense against oxygen toxicity, GPD, is highly stimulated. We found a severe change in the GSH level, showing that myocardial oxidative damage had occurred and also was counteracted at that level.

Studies on animals show that the oxidative stress on reperfusion is correlated with the duration of the ischemic period. Reperfusion after a short period of ischemia (30-60 minutes) does not result in oxidative stress, due to the defense mechanisms still able to protect the myocardial cells against the burst of oxygen free radicals generated by the readmission of oxygen with coronary flow. Reperfusion after a prolonged period of ischemia, when the defense mechanisms are reduced, damages the myocardium, with no recovery in its function.

EVIDENCE OF OXIDATIVE STRESS IN HUMANS DURING POSTISCHEMIC REPERFUSION

Increasing numbers of patients with myocardial ischemia are treated with thrombolytic agents, coronary angioplasty, and coronary artery bypass surgery. There is a growing need to understand the structural and functional events ensuing after re- perfusi0n of ischemic tissue in humans. Clinical evidence of oxidative damage in humans is inadequate due to: (a) the difficulties in following the molecular changes occurring during the early phases of reperfusion; (b) the impossibility of standardizing the

onset, severity, and duration of ischemia and reperfusion; and (c) the lack of reliable indices capable of detecting oxidative stress in humans.

We attemped to resolve this problem by measuring the arterial and coronary sinus difference in GSH and GSSG of 20 CAD patients subjected to different periods of global ischemia followed by reperfusion during coronary artery bypass grafting [18,64]. Due to the high rate of GSH auto-oxidation and its disappearance in the blood, plasma levels of GSH and GSSG were determined using our own modified method [16] in which blood i s immediately treated after collection with thiol reagents: dithionitrobenzoic acid (DTNB) for CSH and N-ethylmaleimide (NEM) for GSSG determination. We have also investigated the relationship between myocardial oxidative stress and functional recovery during postischemic reperfusion in patients with normal ejection fraction and left ventricular and diastolic pressures before the operation.

Reperfusion in patients after a Short period of ischemia (<30 minutes) resulted in a small and transient release in the coronary sinus of GSSG and GSH and in a progressive improvement in hemodynamic parameters, reaching a stable state 4 hours after the operation. In patients with a period of ischemia >30 minutes, reperfusion induced a marked and Sustained release of lactate, GSH, and GSSG; the arteriocoronary sinus difference in GSSG re- mained negative at the end of cardiopulmonary bypass, and the rate of functional recovery was significantly delayed, reaching the values of the patients reperfused early only 12 hours after the operation. In these patients, there was a negative corre- lation between the arteriocoronary sinus difference for GSSG and cardiac output measured 2, 4, and 6 hours after the operation. These data suggest that, depending on the severity of the ischemic period, oxidative stress occurs during reperfusion in patients with CAD subjected to heart surgery and that it may be linked with a delay in postoperative recoverY of cardiac function.

PROTECTION OF MYOCARDIAL FUNCTION DURING ISCHEMIA AND REPERFUSION

The protective mechanisms proposed [65,66] by the interventions used against oxygen toxicity to limit the extent of ischemic and reperfusion damage depend on (a) the inhibition of oxygen free radical production by neutrophils (neutrophil depletion), (b) the enzymatic scavenging of extracellularly produced oxygen free radicals (SOD and catalase) [67- 74], (c) the inhibition of endothelial XO [75], and (d) the free radical scavengers acting both intracellu- larly and extracellularly (ionol, N-2-mercaptopro- pionyl glycine, and dimethylthiourea) [76,77]. We

3C-100S September 30, 1991 The American Journal of Medicine Volume 91 (suppl 3C) -~,


Figure 4. Reduced glutathione (GSH) tissue content after different pharmacologic interventions in vitro. Each compound was administered to the isolated aerobic perfused hearts in the perfusate for i hour at the concentration of 10 -4 /!4. Data of six experiments are presented as mean +_ SEM.

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attempted a new approach to the problem of myocardial protection against oxygen toxicity by increasing the intracellular levels of reduced GSH, being the naturally occurring endogenous scar- enger of oxygen free radicals• A number of different pharmacologic compounds supposedly able to donate SH groups and increase tissue content of GSH were tested. These compounds were added in the perfusate of the isolated and perfused heart (at concentrations ranging from 10 _7 to i0 -4 M) for 1 hour of aerobic perfusion. The heart was isolated and ventricular tissue was homogenized and as- sayed for GSH. The results are shown in Figure 4. The compounds tested were giutathione, S- butyrylglutathione, thioproline, dimercaptopro- panol, captopril, SQ 26703 (a derivative of captopril containing SH groups), and N-acetylcysteine (NAC). Interestingly, only NAC was able to increase GSH content significantly and this effect was dose dependent (Figure 5).

The protective effects of NAC against ischemia and reperfusion damage were studied• NAC is a sulfhydryl group donor, easily transported into the cell where it is deacety!ated and increases the thiol pool, primarily reduced GSH.

When NAC was added to the perfusate 60 minutes before ischemia and continued during ischemia and reperfusion, the rise in diastolic pressure during ischemia and reperfusion was less than that of control hearts and the percentage of recovery in the developed pressure was increased (Figure 6). NAC significantly increased tissue GSH values before the onset of ischemia.

At the end of ischemia, the tissue content of GSH of the hearts pretreated with NAC was significantly higher than that of controls. Pretreatment with NAC had important effects in changes in the cellular redox state occurring on reperfusion. There

was no further reduction in the cellular GSH or increase in the GSSG concentration [78].

The likely explanation for this protective effect is an increase in tissue GSH content after aerobic NAC perfusion (Figure 6). The ischemia-induced GSH decrease was limited by NAC pretreatment, with no GSSG accumulation after reperfusion. This was concomitant with an improved recovery of mechanical function, suggesting that replenishing the reduced thiol pool may be an important development in cellular protection.

However, when NAC was administered after ischemia, at the time of reperfusion, there was little improvement in the recovery of the mechanical function and it did not metabolically protect the isolated hearts. The GSH/GSSG ratio after reperfusion did not change, suggesting that NAC pretreatment is necessary to increase the thiol content of the myocardium and to benefit from its protective effects (Figure 6) [78].

PROTECTION OF HUMAN HEART DURING ISCHEMIA AND REPERFUSION

The positive experimental data on NAC enabled its use at the clinical level. The aim Of the study was to investigate whether NAC, by increasing the cellular thiol pool, reduces the oxidative stress and the functional changes occurring during reperfusion after prolonged periods of ischemia in patients subjected to aortocoronary bypass grafting.

Patients were selected based on their having normal ejection fraction and left ventricular and diastolic pressures before the operation, but coronary artery disease (CAD) at angiographic examination requiring at least four grafts, so that the ischemic period (clamping period) had to be long, more than 30 minutes (mean value of 51.8 -+ 2.2 rain).



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The experimental data showed the need for NAC pretreatment administered at the dosage of 150 mg/kg/hr at i hour before clamping, and at 37.5 mg/ kg/hr during the subsequent 4 hours. NAC was in- fused in the pulmonary artery (through a distal line of a Swan-Ganz catheter) before and after cardiopulmonary bypass, and in the arterial line of the pump during cardiopulmonary bypass. Control patients received tha same amount of saline administered with the same modality.

The occurrence of oxidative stress was assessed by measuring the formation and release of GSSG and reduced GSH in the coronary sinus. Standard hemodynamic measurements were recorded during the 24 hours after cardiopulmonary bypass.

So far we have studied only six patients and the results of two selected cases (one control and one treated) having identical coronary anatomy, preop- erative hemodynamics, and clamping periods, are shown in Figures 7 and 8. As expected, before clamping, both patients showed no arteriocoronary

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Figure 5. Dose-response effects of N-acetyi- cysteine (NAC) on tissue content of reduced glutathione (GSH) and oxidized glutathione (GSSG). The modalities of the experiments are identical to those described in Figure 4. Data are presented as mean +- SEM of five experiments.

sinus difference in GSSG. Reperfusion of the untreated patient showed marked and sustained release of GSSG, continuing after the end of cardiopulmonary bypass (Figure 7). The rate of functional recovery after the operation was slow, reaching a steady state only 12 hours after the operation (Fig- ure 8).

In the patient pretreated with NAC, reperfusion showed only a small and transient release in GSSG in the coronary sinus, followed by a prompt ira- provement of hemodynamic parameters, reaching a stable state 5 hours after the operation (Figures 7 and 8).

All other patients showed similar behavior, although they also showed a greater degree of vari- ability.

In two patients, hypotension occurred at the end of cardiopulmonary bypass, with one requiring pharmacologic treatment.

These preliminary data suggest that NAC could exert in humans the same cardioprotective effect

3C-102S September 30, 1991 The American Journal of Medicine Volume 91 (suppl 3C)


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Figure 8. Recovery of cardiac output in the 24 hours following coronary artery bypass in the two typical examples reported in Figure 7. CPB: cardiopulmonary bypass.

demonstrated in experimental studies. However, there is a need to investigate the correct dosage before its wide application; it could, at high dos- ages, cause hypotension.

If the preliminary data on NAC treatment were verified in a large study, they could be of impor- tance, as NAC represents a completely different protective method compared with the traditional drugs such as calcium antagonists and/~-blockers, acting by influencing the inotropic state of the myocardium.

ACKNOWLEDGMENT This work has been supported by the C.N.R. Target Project on Biotechnology and Bioinstrumentation and by the C.N.R. Project FAT.MA.

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oxygen free radicals and myocardial damage: protective role of thiol-containing agents

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