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Ann Thorac Surg 1995;60:1289-1293
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Superoxide Radical and Xanthine Oxidoreductase Activity in the Human Heart During Cardiac Operations

Departments of Cardiac Surgery and General Surgery, Mater Misericordiae Hospital, Dublin, Ireland

Accepted for publication June 16, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. The results of clinical trials of xanthine oxidoreductase inhibition in cardiac surgery are encouraging, although studies have failed to localize the enzyme to the human heart and to localize free radical activity to fresh human heart.

Methods. We adapted a histochemical staining technique based on the reduction of nitro blue tetrazolium to formazan by superoxide radical. In six samples of right atrium graded blindly on a scale of 0 through 4, strong staining (median grade, 3) of the microvasculature was seen. This was blocked by allopurinol in paired sections (median grade, 1; p < 0.01). Chemiluminescence can be used as an index of superoxide radical activity. Atrial samples were taken from 13 patients at five time points during coronary bypass grafting and placed in buffered luminol. Then chemiluminescence was measured.

Results. A 15-fold rise in chemiluminescence (295.93 ± 39.47 mV) was demonstrated during reperfusion compared with the control value (19.06 ± 0.47 mV). Chemiluminescence at 1 minute after release of the cross-clamp was significantly higher (p < 0.05) by analysis of variance versus values obtained before bypass and 1 minute before and 30 minutes after reperfusion.

Conclusions. In this study we have identified superoxide radical activity and a possible generating system (xanthine oxidoreductase) in the human heart.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Free radical–mediated tissue injury is now recognized as a final common pathway of damage in a wide variety of pathophysiologic processes. The xanthine oxidoreductase–based free radical–generating mechanism has been found to operate in many different organ systems. In this mechanism, hypoxanthine and xanthine accumulate during ischemia from the breakdown of purines. Concomitantly, ischemia converts the nonradical-generating enzyme xanthine dehydrogenase to the radical-generating form xanthine oxidase. On the reintroduction of oxygen at reperfusion, a burst of superoxide radical is generated and causes the subsequent tissue injury [1]. The entire xanthine oxidase–based free radical–generating system is present and operative in the endothelial cell [2].

The role of oxygen-derived free radicals in ischemia-reperfusion injury to the human heart during a routine cardiac operation is unclear. Definitive evidence is lacking, although routine cardiac procedures with isolation of the heart by cross-clamping the aorta would seem to be an ideal environment for superoxide radical production. Several studies [3, 4] have shown an increase in lipid peroxidation products (presumed as a result of free radical injury) in the systemic circulation during cardiac operations but did not localize the generating organ or system.

Clinical studies [5–7] in patients having coronary artery bypass grafting and receiving oral doses of allopurinol (a competitive inhibitor of xanthine oxidoreductase) have shown a beneficial effect. This is presumed due to extracardiac inhibition of xanthine oxidase, as studies [8, 9] have failed to detect xanthine oxidoreductase (dehydrogenase plus oxidase) activity in the human heart. In contrast, there is evidence from immunofluorescence techniques that xanthine oxidase is present in the capillary endothelium of human heart muscle [10].

Luminol-enhanced chemiluminescence of tissue samples can be used as a direct index of oxygen-derived free radical activity [11] and gives real-time information on the extent of this activity at any given point [12]. In experimental studies [13, 14], the technique has demonstrated an increase in chemiluminescence in rabbit and rat myocardium during ischemia-reperfusion–induced cardiac injury.

Histochemical staining can be used to localize xanthine oxidoreductase activity. The technique is a modification of the method of Auscher and colleagues [15] (used in rat liver and jejunum), the basis of which is the reduction of nitro blue tetrazolium by xanthine oxidoreductase into insoluble formazan in phosphate buffer with hypoxanthine as substrate. The resulting purple stain localizes enzyme activity.

The aim of this study was to detect superoxide radical activity in samples of human right atrium during reperfusion in routine cardiac operation and to determine if the superoxide radical–generating enzyme xanthine oxidoreductase is present in the same tissue samples. We think this is a fundamental step in understanding the mechanism and possible therapeutic manipulation of ischemia-reperfusion injury to the human heart.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Patient Population
Thirteen patients scheduled for routine coronary artery bypass grafting were included in the study. Informed consent was obtained from every patient. There were 9 men and 4 women. Age ranged from 47 to 71 years (mean age, 61.3 years).

Operative technique was similar in all patients. After standard aortic and right atrial cannulation, the patient was systemically cooled to an arterial blood temperature of 28°C, the aorta was cross-clamped, and 1 L of St. Thomas' cardioplegic solution was administered by antegrade infusion. If the cross-clamp time was anticipated to be greater than 45 minutes, a further dose of 500 mL of cardioplegia was infused every 30 minutes. All patients had a left internal mammary artery–left anterior descending coronary artery graft. All other grafts were saphenous vein. Proximal vein graft anastomoses were performed after removal of the aortic cross-clamp. Mean cardiopulmonary bypass time was 84 ± 5 minutes; mean cross-clamp time was 47 ± 4 minutes; and the median number of grafts was three.

Chemiluminescence Protocol
Luminescence is the emission of light by a nonthermal process. In the case of chemiluminescence, the light is produced by a chemical reaction. The molecules responsible for emitting the light absorb free energy released by a chemical reaction, thereby raising electrons to a higher energy level. These electrons return to the more stable lower energy state and emit light. The intensity of the light emitted depends on the reaction rate, which, in turn, depends on the concentration of the molecules taking part in the reaction. Light intensity is therefore directly proportional to the concentration of the reactants. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a synthetic compound that emits light at 425 mm when oxidized by superoxide radical. This light is detected by the photomultiplier tube in the chemiluminometer.

From all 13 patients, samples (approximately 1 mm³) were taken from right atrial appendage muscle not encircled by the venous cannulation pursestring. In initial experiments in which samples were taken from atrial muscle within the pursestring (an area that remains ischemic after release of the aortic cross-clamp), no chemiluminescence activity could be demonstrated.

Samples were immediately placed (without any prior treatment) in a reaction solution containing 400 µL of buffer (phosphate-buffered saline solution, 1 mmol/L HEPES buffer, 5 mmol/L glucose), 40 µL of luminol (2 mg/mL in phosphate-buffered saline solution), and 20 µL of 30% triethylamine. Chemiluminescence was measured with a 1250 Wallac LKB luminometer (LKB, Stockholm, Sweden). The stable peak chemiluminescence value (usually within 1 minute) was recorded, each sample was weighed, and peak chemiluminescence activity (mV) per 100 mg of tissue sample was calculated. The atrial biopsy site was oversewn after the last sample had been collected. Samples were taken at five time points: before bypass (control); before removal of the cross-clamp; 1 minute after cross-clamp removal; 15 minutes after cross-clamp removal; and 30 minutes after cross-clamp removal.

The luminometer was calibrated using increasing concentrations of hydrogen peroxide (Fig 1). The relationship over the range measured was linear.

View larger version (18K): [in this window] [in a new window]   Fig 1. . Chemiluminometer calibration curve. The relationship between increasing concentrations of hydrogen peroxide and peak chemiluminescence is linear.

Specimens were graded blindly by a single observer on the amount of blue or purple staining seen microscopically (grade 0 = no staining, grade 4 = strongest staining seen).

Statistical Analysis
Apparent differences between samples were evaluated for statistical significance using repeated-measures analysis of variance for the parametric data (chemiluminescence). As the variations in the standard deviations between the groups were large, the data were transformed to the square root value of peak chemiluminescence ± the standard deviation before statistical analysis. The Wilcoxon signed-rank test was used for nonparametric data (grades of staining).

Parametric values are expressed as the mean ± the standard error of the mean. Nonparametric values (grades of staining) are expressed as the median value for each group. The p values that were 0.05 or less were considered significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Chemiluminescence
Peak chemiluminescence activity per 100 mg of tissue was measured at each of the five time points. The control value before bypass of 19.06 ± 0.74 mV rose to 45.17 ± 3.0 mV 1 minute before release of the cross-clamp. During the reperfusion phase at 1 minute after release of the cross-clamp, a 15-fold rise (295.93 ± 39.47 mV) in chemiluminescence compared with the control value was measured. This subsequently declined toward the control level with values of 223.06 ± 20.79 mV at 15 minutes and 63.02 ± 12.38 mV at 30 minutes after release of the cross-clamp. The chemiluminescence level at 1 minute after release of the cross-clamp was significantly higher (p < 0.05) than the values obtained before bypass; and 1 minute before and 30 minutes after release of the cross-clamp. The chemiluminescence level at 15 minutes after removal of the cross-clamp was significantly higher (p < 0.05) than the levels seen before bypass and 30 minutes after release of the cross-clamp (Fig 2).

View larger version (21K): [in this window] [in a new window]   Fig 2. . Chemiluminescence activity in human myocardium (n = 13) during cardiac operations. The square root values of peak chemiluminescence are plotted against the five time points of the experiment. (CCO = cross-clamp off; p 0.05 versus before bypass, before CCO, and 30 minutes after CCO; ¶ p 0.05 versus before bypass and 30 minutes after CCO.)

View larger version (12K): [in this window] [in a new window]   Fig 3. . Histochemical staining for xanthine oxidoreductase in the human heart (n = 6 patients). Each dot represents an individual staining grade. Specimens incubated in the hypoxanthine medium are shown on the left with increasing staining grades on the horizontal axis, and specimens incubated with allopurinol added to the medium are shown on the right. The graphs are significantly different (p 0.01 by Wilcoxon signed-rank test). (* = median grade.)

View larger version (149K): [in this window] [in a new window]   Fig 4. . Photomicrograph of human heart (right atrial appendage) stained for xanthine oxidoreductase showing typical dark (blue) color predominantly localized to the blood vessels. (Nitro blue tetrazolium; x10 before 48% reduction.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

A free radical is a molecule with one (or more) highly reactive unpaired electron in its outer orbital. Free radicals are highly reactive particles that can attack any biochemical component of a cell, but lipids, proteins, and nucleic acids are the most vulnerable. The most common radical in biologic systems is molecular oxygen, which has two unpaired electrons. Other important oxygen-derived free radicals are the superoxide anion, the hydroxyl anion, and alkoxyl and peroxy radicals. Other free radicals, such as transition metal ions, atomic hydrogen, carbon, and sulfur-centered radicals, also play an important secondary role [16].

These toxic metabolites of oxygen have been implicated in a number of diseases, to date, mainly in the gastrointestinal tract. Many oxidases are capable of generating oxygen-derived free radicals, but attention has focused on xanthine oxidase in various organs as the source of superoxide radical–mediated ischemia-reperfusion injury. Under normal conditions, the enzyme is involved in the metabolism of purines by catalyzing the two-step oxidation of hypoxanthine through xanthine to urate. In vivo xanthine oxidoreductase (the full name of the enzyme) exists in one of two forms, the dehydrogenase or d-form (which donates the electrons removed from the substrate to NAD⁺ [nicotinamide adenine dinucleotide, oxidized form]) or the oxidase or o-form (which donates electrons to molecular oxygen, thus generating the superoxide radical anion). Therefore, although either form will oxidize hypoxanthine to uric acid, only the oxidase (o-form) generates a free radical as a by-product.

Granger and colleagues [1] first proposed the mechanism for xanthine oxidase–mediated reperfusion injury. During ischemia, the breakdown of adenosine triphosphate to hypoxanthine provides substrate for xanthine oxidase, while at the same time, the dehydrogenase form of the enzyme is converted to the oxidase form. As a result, the enzyme produces a burst of superoxide as hypoxanthine is oxidized when oxygen is reintroduced, and this causes the subsequent tissue damage [2]. The injury can be blocked by inhibiting the generation of superoxide with allopurinol (a competitive inhibitor of xanthine oxidase) or scavenging the oxygen-derived free radicals produced with superoxide dismutase, catalase, or both.

We believe that this mechanism of ischemia-reperfusion injury operates in the human heart. Oxygen-derived free radicals generated from activated xanthine oxidase located in the microvasculature would initiate a capillary endothelial cell injury leading to myocardial instability.

Evidence supporting this hypothesis in humans is inconclusive, to date. Two studies [8, 9] have failed to demonstrate measurable levels of the free radical–generating enzyme xanthine oxidase in homogenized human heart. It is possible that by homogenizing heart tissue, small, specifically localized quantities of the enzyme may have been obscured. Endothelial cells (comprising < 1% of myocardial tissue) represent such a specific site. Immunofluorescence techniques with polyclonal antibodies suggest that xanthine oxidase is present in the capillary endothelium of human heart muscle [10].

There is also little direct evidence of superoxide radical generation during cardiac operations. Paired frozen left ventricular samples were used to assess oxidative stress in a group of 6 patients undergoing coronary artery bypass grafting [17]. A twofold increase in chemiluminescence was measured between samples taken before and after cross-clamp release. Right ventricular biopsy specimens were analyzed for hydroxy-conjugated dienes (a chemical marker of free radical injury) in 6 cyanotic children during repair of tetralogy of Fallot [18]. Although elevated compared with canine controls, no difference was seen in diene levels during the operations. More elaborate methods of measuring oxygen-derived free radicals such as electron spin resonance are suitable only for in vitro experiments.

Lipid peroxidation products can be measured in peripheral or coronary sinus blood as a crude indirect marker of free radical damage. These assays are nonspecific in that neither the source of generation nor the type of free radical is identified. Several studies in coronary artery bypass patients have shown either an increase in lipid peroxidation products after release of the cross-clamp [3, 5] or after cessation of bypass [4].

Chemiluminescence has been used to assess oxygen-derived free radical activity in an experimental heart preparation [13], rat colon carcinoma [19], and lung injury during cardiac surgical intervention in humans [20]. The technique we describe is easily reproducible, is simple and safe to perform, and requires little ancillary equipment. Our modification of the chemiluminescence technique analyzes samples of fresh human right atrium unlike all previous studies, which analyzed supernatants from frozen myocardial samples. We believe this should more accurately reflect oxygen-derived free radical activity in a tissue sample at any given time point. It is unclear whether chemiluminescence represents superoxide radicals or the resultant secondary radicals and their breakdown products. The LKB luminometer is fully portable and can be used in or close to the operating room. As such, it is highly suitable for studying superoxide radical activity during operations. This technique should be a useful tool in the clinical assessment of ischemia-reperfusion injury during cardiac surgical procedures.

Our initial experiment shows the relationship of increasing concentrations of hydrogen peroxide and chemiluminescence over the range measured to be linear. Subsequently, we found that compared with the baseline value, there was a 15-fold rise in right atrial chemiluminescence 1 minute after the aortic cross-clamp was removed, which declined over the next 30 minutes. This is entirely in keeping with the original theory of ischemia-reperfusion injury [21]. On the reintroduction of oxygen into the ischemia-primed system, there is a burst of superoxide radical production, which subsequently declines depending on the ischemic time. We have not determined in this study whether the source of the superoxide radical generation is activated xanthine oxidase, neutrophils, or a combination of both. In future studies with this technique, we hope this will become apparent. We intend to apply the technique to the assessment of therapeutic manipulation of ischemia-reperfusion injury to the heart during coronary bypass grafting.

Xanthine oxidoreductase enzymatic activity has been localized using histochemical techniques to the rodent liver [15], duodenum [22], muscle fibers, enterocytes, hepatocytes, and renal collecting tubules [23]. This histochemical technique is based on the reduction of the yellow-colored nitro blue tetrazolium to the insoluble blue precipitate formazan in phosphate buffer with hypoxanthine as substrate. This should occur only if the enzyme xanthine oxidoreductase is present in the medium, usually in the form of tissue sections. Under aerobic conditions if NAD is added to the reaction, both the dehydrogenase and oxidase forms of the enzyme will stain. The advantage of this method over other localization techniques is that it specifically localizes enzyme activity, thus allowing small quantities of enzyme to be detected. Other oxidoreductase enzymes will also reduce nitro blue tetrazolium, and this is controlled by the use of allopurinol.

The technique has been further developed to localize xanthine oxidoreductase activity in the rat pancreas, another organ in which previous attempts at localization have been unsuccessful because of a nonhomogeneous distribution and the confounding effect of resident proteases [24]. Further, in rat liver and small bowel (organs known to be rich in xanthine oxidoreductase activity), the intensity and tissue specificity seen correlated precisely with immunohistoaffinity staining and were completely blocked by allopurinol, showing specificity for xanthine oxidoreductase. The histochemical staining of uniform sections of rat liver, quantitated by light absorption at 570 nm, correlated precisely with levels of xanthine oxidase activity by enzymatic assay of homogenates of paired specimens. Incubation with and without NAD allowed quantitative discrimination of the free radical–generating oxidase from the dehydrogenase form and also correlated precisely with enzymatic assay in homogenates of paired specimens [25].

As the level of xanthine oxidoreductase activity in humans is considerably less than in rodents, we concentrated on staining for total enzymatic activity (dehydrogenase and oxidase) rather than attempting to differentiate between the two forms. Staining of samples of human heart showed xanthine oxidoreductase activity to be located chiefly in the microvasculature. Again, this is in keeping with the theory that the entire xanthine oxidoreductase–generating system is within the endothelial cell. We hope to further develop this technique to allow us to study both xanthine oxidase activity and rate of conversion of dehydrogenase to oxidase.

This study demonstrates that superoxide radical activity is detectable by chemiluminescence in samples of human right atrium during the reperfusion phase of routine coronary artery bypass grafting. The superoxide radical–generating enzyme xanthine oxidoreductase is also detectable in the same tissue samples. Although the study was limited to right atrial samples, we have identified superoxide radical activity and a possible generating system in the human heart.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work was funded by a Mater Hospital College for Postgraduate Education and Research Grant.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Mr MacGowan, Department of Cardiac Surgery, Mater Misericordiae Hospital, Eccles St, Dublin 7, Ireland.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Simon W. MacGowan Alfred E. Wood Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MacGowan, S. W. Articles by Wood, A. E. Search for Related Content PubMed PubMed Citation Articles by MacGowan, S. W. Articles by Wood, A. E.