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Ann Thorac Surg 1995;59:169-172
© 1995 The Society of Thoracic Surgeons

Deferoxamine Cardioplegia Reduces Superoxide Radical Production in Human Myocardium

Ahepa General Hospital, University of Thessaloniki, Thessaloniki, Greece

Accepted for publication August 8, 1994.

	Abstract

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
Recent studies have demonstrated enhanced myocardial protection during ischemia using the oxygen free radical scavenger, deferoxamine. This effect of deferoxamine may be related either to its iron-chelating property or to intervention in an iron-independent mechanism. We tested the latter by determining the rate of superoxide anion production and the degree of lipid peroxidation in human myocardial tissue after including deferoxamine in cardioplegic solution. Fourteen patients who underwent aortic, mitral, or double valve replacement were included in the study. The mean value for superoxide radical production was 59.8 ± 17.0 nmol • min^-1 • g^-1 for the control group (group C; n = 7) and 21.3 ± 8.1 (p < 0.001) for the deferoxamine-treated group (group D; n = 7). The mean value for thiobarbituric reactive substances was 80.00 ± 23.4 in group C and 38.7 ± 23.8 nmol • min^-1 • g^-1 in group D (p < 0.01). In conclusion, deferoxamine appears to have a moderating effect on the biochemical markers of ischemia reperfusion injury. Its scavenging effect on superoxide anion could play a role in the cellular defense against oxygen radicals during cardiac operations.

	Introduction

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
Improvements in the methods for myocardial protection over the past 10 years have contributed to a reduction in operative mortality. In this context, there is increasing experimental evidence that reactive oxygen species are involved in the pathogenesis of myocardial tissue injury associated with ischemia and reperfusion [1, 2].

The Haber-Weiss and Fenton reactions produce the most toxic oxygen species, the hydroxyl radical (^•OH). This is readily formed in biologic systems in the presence of transition metals, particularly iron, which catalyzes the two-step Fenton reaction. This is summarized in the following reactions:

(1)

(2)

This propagation sequence of radical-generating reactions has important biological effects because one of the targets for free radical injury is the lipid component of cell membranes [3].

Recent investigations have demonstrated enhanced myocardial protection by the use of oxygen free radical scavengers [4, 5]. In particular, it has been proposed that chelation of the ferric iron with deferoxamine can reduce the production of the hydroxyl radical, thereby moderating myocardial ischemia-reperfusion injury [6, 7]. This effect of deferoxamine may be related either to its iron chelating property or to intervention in an iron-independent mechanism.

Although deferoxamine has been used to scavenge the production of hydroxyl radical [8], there is evidence that it can also react directly with superoxide radicals [9]. Furthermore, in vitro preparations have demonstrated the ability of deferoxamine to inhibit lipid peroxidation [10]. The purpose of the present study was to test the ability of deferoxamine to scavenge superoxide radicals. We investigated this by determining the rate of superoxide anion production in human myocardial tissue after including deferoxamine in the cardioplegic solution. In addition, we determined the degree of lipid peroxidation in the same samples as an index of free radical activity.

	Patients and Methods

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
The study was first approved by the ethics committee of Ahepa General Hospital.

Fourteen patients who underwent elective, first-time operations on the aortic, mitral, or both valves were included in the study. After informed consent, they were randomly allocated to either standard St. Thomas' cardioplegic solution (group C, n = 7) or cardioplegic solution supplemented with deferoxamine (Desferal; Ciba-Geigy), 1,000 mg/L (group D, n = 7). The surgical procedures are listed in Tables 1 and 2. The ages in group C ranged from 51 to 71 years (mean, 59.1 years) and in group D from 50 to 76 years (mean, 62 years).

Cardiopulmonary bypass was performed with a roller pump and a Cobe CML membrane oxygenator at a flow rate of 2.2 L • m^-2 • min^-1. A single venous cannula was used for aortic valve replacement and separate caval cannulas were used for mitral and double valve replacements. The perfusate was cooled to 28°C. Topical hypothermia with crushed-ice saline was added. The composition of the crystalloid cardioplegia was as follows: Na, 147 mmol/L; K, 20 mmol/L; Ca, 2 mmol/L; Cl, 203 mmol/L; Mg, 16 mmol/L; procaine, 1%; and osmolarity, 388 mOsm.

The cross-clamp time for group C ranged from 35 to 102 minutes (mean, 56.71 minutes) (see Table 1) and for group D it ranged from 32 to 120 minutes (mean, 72.7 minutes) (see Table 2). In both groups, 1,000 mL of appropriate cardioplegic solution was administered into the aortic root at a temperature of 4°C and a pressure of 100 mmHg. After the initial 30 minute cross-clamp period, an additional 200 to 500 mL of cardioplegic solution was infused.

Myocardial biopsy specimens were obtained from the apex of the left ventricle 10 minutes after aortic cross-clamp release. The specimens were obtained using a size 14 scalpel blade and they were immersed immediately in liquid N₂ and stored at -70°C.

Determination of Superoxide Radical Production
The rate of superoxide anion generation was determined by measuring the absorption at a wavelength of 500 nm during the reduction of ferricytochrome C [11]. Each sample was placed into a polystyrene tube containing 1 mL of Krebs bicarbonate buffer (in mmol/L: NaCl, 118; KCl, 4.7; MgSO₄, 1.2; NaHPO₄, 1.2; NaHCO₃, 25; and glucose, 11). Then 15 µmol/L cytochrome C (type VI from horse heart) was added immediately and the sample was incubated for 15 minutes in a water bath at 37°C. At the end of this period, the buffer was removed and the absorbance was read at 550 nm after the addition of 3 mmol/L N-ethylmaleimide to inhibit further reduction of cytochrome C. One milliliter of new buffer was added to the sample, followed by the addition of 3 mmol/L N-ethylmaleimide. Then 15 µmol/L cytochrome C was added and the incubation was continued for another 15 minutes. This final mixture was used as a blank for the same sample of tissue.

The amount of O₂^- produced was calculated by dividing the absorbance of the sample by the extinction coefficient for the change between ferricytochrome c and ferrocytochrome c, E₅₅₀ = 21 nmol/L • cm^-1, and the results were expressed in nanomoles of O₂^-/min per gram.

Determination of Lipid Peroxidation
The amount of lipid peroxidation was assessed by determining the tissue content of thiobarbituric acid reactive substances [12]. Tissue was homogenized in a reagent containing 15% wt/vol trichloroacetic acid, 0.375% wt/vol thiobarbituric acid, and 0.25 N hydrochloric acid. After the addition of 0.01% butylated hydroxytoluene to prevent further lipid peroxidation, the homogenate was heated for 15 minutes in a boiling water bath. After cooling, the precipitate was removed by centrifugation. The absorbance of the supernatant was measured at 535 nm against a blank that contained all the reagents except the lipid. The concentration of the thiobarbituric acid reactive substances was calculated using the extinction coefficient of 1.56 x 10⁵ mol/L • cm^-1 and results were expressed in nanomoles of thiobarbituric acid reactive substances per minute per gram of tissue.

Statistical Analysis
All values were expressed as the mean ± standard deviation. Statistical significance was defined as p less than 0.01 using a Student's nonpaired t test.

	Results

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
Superoxide Radical Production
The data for superoxide anion production are shown in Tables 1 and 2. The generation of superoxide radicals was significantly lower in patients treated with deferoxamine (group D) than in the control group. The mean value was 59.8 ± 17.0 nmol/min per gram for the control group (n = 7) and 21.3 ± 8.1 nmol/min per gram for group D (n = 7). The two values were significantly different as assessed by Student's t test (p < 0.001).

Determination of Lipid Peroxidation
Measurement of thiobarbituric acid reactive substances in samples of groups C and D showed that deferoxamine significantly inhibited lipid peroxidation. The mean value for these substances was 80.0 ± 23.4 in group C (n = 7) and 38.7 ± 23.8 in group D (n = 7) (p < 0.01), as assessed by Student's t test.

	Comment

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
At least two different mechanisms are known to result in free radical production during cardiopulmonary bypass. First, it is established that cardiopulmonary bypass results in a complement-mediated activation of neutrophils [13]. The neutrophils in turn generate oxygen free radicals, in particular superoxide anions and hydrogen peroxide [14]. Second, most cardiac operations involve a transient period of aortic cross-clamping. This ischemia-reperfusion sequence results in generation of free radicals in the myocardium [15]. Ischemia also reduces the activity of the enzymatic cell defenses against free radicals. On reperfusion, the delicate balance of oxidants/antioxidants is further disturbed by introducing oxygen and generating a burst of free radical activity [16]. The free radical reactions are catalyzed by chelated forms of iron. Because these are low-affinity chelates, one therapeutic approach is to provide a drug that will form a very high-affinity, catalytically inactive chelate. Deferoxamine mesylate, which is an effective iron chelator capable of entering the intracellular space [17], is usually selected for this purpose. The drug is specific for chelating iron and is currently used to treat patients who are in an iron-overload state. It has been reported that deferoxamine prevents superoxide-dependent iron reduction and inhibits the iron-catalyzed generation of hydroxyl radicals as well as lipid peroxidation [18]. An additional advantage of deferoxamine could be its ability to act as a direct scavenger of some activated oxygen species [9]. In this context, we tested the ability of deferoxamine to scavenge superoxide radicals in patients undergoing open heart operations. Our results show that generation of superoxide anion as well as lipid peroxidation were significantly lower in patients treated with deferoxamine.

We gave deferoxamine as an additive to cardioplegic solution in a dose of 1,000 mg/L. This is particularly important because greater doses of the drug have a prooxidant action. The dose of deferoxamine delivered to the myocardium was similar to that used by Fereira and associates [19] and by Menasché and colleagues [18].

Previous reports of the use of deferoxamine in myocardial preservation have yielded conflicting results. In vitro studies suggest that deferoxamine improves membrane stability and decreases creatine kinase release after ischemia [20]. In addition, there is a significant reduction in arrhythmias and an increased incidence of sinus rhythm during reperfusion when deferoxamine was present in the perfusate [21]. Furthermore, Ambrosio and co-workers [22] showed that administration of deferoxamine at the time of postischemic reflow resulted in improved recovery of myocardial function and energy metabolism. Conversely, in an animal model of regional ischemia, Bolli and associates [23] noted no changes in regional blood flow or ventricular function. Similar results were reported in a rabbit model by Maxwell and associates [24]. In a recent report de Boer and Clark [25] raised the question of the optimum timing of iron chelation therapy with respect to ischemia and the dose–response relationships of deferoxamine for improved myocardial protection. They showed that pretreatment with deferoxamine significantly improved survival and they concluded that deferoxamine given after the ischemia insult was of little value [25].

Menasché and associates [18] used deferoxamine both intravenously and as an additive to the cardioplegic solution in a group of patients undergoing cardiopulmonary bypass. They found that, after bypass, neutrophils harvested from deferoxamine-treated patients produced significantly fewer superoxide radicals than those of control patients. As might be expected in the current small study group, the clinical outcome of the control and treated patients was the same. There was no difference in hemodynamic status, dependence on inotropic agent support, or postoperative arrhythmias between the two groups. Similarly, Fereira and colleagues [19] did not observe any improvement in clinical outcome or rate of perioperative infarction, although there was a better preservation of myocardial cells in specimens taken from deferoxamine-treated patients. Further, more extensive studies of clinical outcome are indicated with specific reference to operative mortality, hemodynamic parameters, and need for inotropic agent support in matched patient groups randomized to deferoxamine cardioplegia or placebo.

Deferoxamine in cardioplegic solution reduced myocardial oxidative stress, as shown by a marked decrease in the generation of superoxide anion in myocardial tissue as well as a decrease in lipid peroxidation products. Our results show that deferoxamine can inhibit superoxide anion generation, a finding consistent with previous study of Sinaceur and associates [9], who claim that Desferal reacts directly with the O₂^- radical.

In conclusion, deferoxamine has a moderating effect on the biochemical markers of ischemia-reperfusion injury. Whether this scavenging effect on both superoxide anion and OH radical could have a clinical role in the cellular defense against oxygen radicals during cardiac operations should be investigated further.

	Footnotes

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 
Address reprint requests to Dr Drossos, Ahepa General Hospital, University of Thessaloniki, Thessaloniki, Greece.

	References

Top Footnotes Abstract Introduction Patients and Methods Results Comment References

 

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