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Ann Thorac Surg 1995;60:1735-1740
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Blood Cardioplegia Increases Plasma Iron Overload and Thiol Levels During Cardiopulmonary Bypass

Departments of Cardiothoracic Surgery and Anaesthesia & Intensive Care, Royal Brompton Hospital, National Heart & Lung Institute, London, United Kingdom

Accepted for publication August 2, 1995.

	Abstract

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Background. Cardiopulmonary bypass and cross-clamping of the ascending aorta introduce two well-characterized phases of oxidative stress, namely, the extracorporeal circulation of blood and the reoxygenation of ischemic tissue. A feature of both forms of stress is the release of reactive and damaging oxygen species.

Methods. Forty-seven patients undergoing aortic valve replacement received either cold crystalloid, cold blood, or warm blood cardioplegia. Plasma thiol levels were measured in all groups before and during bypass. All cardiopulmonary bypass patients had, before going onto bypass, low plasma thiol levels (3.80 ± 0.22 nmol/mg protein) compared with normal healthy controls (5.48 ± 0.14 nmol/mg protein).

Results. Thiol values remained low throughout bypass in patients receiving cold crystalloid cardioplegia, but rose in patients receiving cold blood cardioplegia, and rose even more in patients receiving warm blood cardioplegia to reach normal plasma values. During cardiopulmonary bypass it has previously been reported that plasma transferrin can become fully saturated with iron and cause transient iron overload. Two patients (13%) receiving cold crystalloid cardioplegia went into plasma iron overload, whereas 18% receiving cold blood and 27% receiving warm blood cardioplegia showed plasma iron overload.

Conclusions. We suggest that blood cardioplegia provides an additional source of thiols as well as a source of reactive iron. However, the reactive iron and thiol-containing molecules have the potential to interact and exacerbate oxidative stress, already a feature of bypass. Control of reactive iron by chelation may be strongly indicated when blood cardioplegia is used.

	Introduction

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See also page 1740.

Circulation of blood extracorporeally through plastic tubing causes severe shear stresses to blood cells and activates several regulatory cascades. As a result, a situation of oxidative stress ensues as red blood cells are lysed, and neutrophils are ``activated'' to produce superoxide and hydrogen peroxide [1]. Hydrogen peroxide can react with hemoglobin to release redox active forms of iron from the heme moiety [2], able to participate in organic and inorganic oxygen radical reactions by stimulating lipid peroxidation and catalyzing the formation of the highly reactive and damaging hydroxyl radical. Iron that is not safely sequestered in the body is therefore a serious risk factor for oxidative damage. In certain patients undergoing cardiopulmonary bypass the release of iron is so great that it can saturate plasma transferrin and lead to transient plasma iron overload [1, 3]. In most cardiopulmonary bypass patients, however, iron release is minimal and leads to an increased iron loading of transferrin achieving around 50% saturation [3]. The iron-binding capacity of transferrin provides a potent antioxidant potential against iron-driven free radical reactions [4], and part of this protection is lost in most cardiopulmonary bypass patients [5]. When the plasma transferrin becomes fully saturated with iron all iron-binding antioxidant activity is lost and plasma shows prooxidant properties.

Reperfusion injury, a feature of cardiopulmonary by-pass procedures, causes an additional phase of oxidative stress, and this is greatly exacerbated if reactive forms of iron are present [6]. Reoxygenation injury, on reperfusion of cardiac tissue, is thought to arise through the multifactorial generation of free radicals and other reactive oxygen species triggered by biochemical changes occurring during the period of ischemia [reviewed in 7].

Normal human plasma contains around 500 µmol/L of thiols, and these are predominantly protein sulphydryls mainly associated with albumin. The low molecular mass thiol molecule glutathione (GSH) is only a minor plasma constituent, accounting for 2 µmol/L or less of total plasma thiols. Glutathione is an intracellular molecule and plays a pivotal role in maintaining the body's thiol pool, in which there is a constant exchange between protein thiols, reduced and oxidized glutathione, and mixed disulfides. Changes in plasma thiols may, to some extent, reflect changes in tissue thiols resulting from oxidative stress.

Recent studies in patients with acute lung injury have shown that plasma protein thiol levels are good predictive markers of morbidity and mortality [8]. Loss of plasma thiols parallels other markers of free radical damage to proteins such as carbonyl formation [8] and peroxidation of lipids with formation of 4-hydroxy-2-nonenal [9]. The patients studied here were prospectively randomized into three groups depending on the type of cardioplegia they received. The first group received cold crystalloid cardioplegia, the second cold blood cardioplegia, and the third warm blood cardioplegia.

	Material and Methods

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This prospective study involved 47 patients (32 men; age range, 24 to 82 years) undergoing aortic valve replacement, with or without artery grafting. Operation was carried out by a single surgeon. All blood samples (10 mL) were collected from a cannula in the coronary sinus into lithium heparin tubes, stored at 4°C, and transported to the laboratory at the end of operation for immediate separation and analysis. Blood samples were taken at the following time points: chest open (prebypass control sample), at the start of bypass, at the end of the ischemic period, at the end of bypass, and off bypass before administration of protamine. The bypass circuit was primed with 1.5 L of Hartman's solution, and the patient was cooled to 25°C. Institutional Ethical Committee approval was obtained for this study. Venous blood samples were also taken from 24 normal healthy controls (mean age, 31 years).

Patients were randomized in a blind, prospective manner for the method of myocardial protection. Group A (15 patients; 8 male; mean age, 58 years) received cold crystalloid St. Thomas No. 1 solution cardioplegia in an antegrade manner. The initial dose was 150 mL/m² body surface area per minute for 3 minutes at 4°C (approximately 900 mL). Subsequent maintenance doses were 150 mL/m² body surface area (approximately 300 mL) given over 1 minute. All doses in group A were given by direct coronary cannulation.

Group B (17 patients; 11 male; mean age, 60 years) received cold blood cardioplegia. The formulation was similar to that for group A except that the patient's own blood was added to the St. Thomas No. 1 solution in a ratio of 4:1 (blood:crystalloid). The initial and maintenance doses were identical to group A but the route of administration differed. For the initial dose, two thirds was given antegrade by direct cannulation of the coronary artery, and one third was given retrograde into the coronary sinus. All subsequent maintenance doses were given by the retrograde route.

Group C (15 patients; 13 male; mean age, 59 years) received warm blood cardioplegia. The formulation and the delivery were identical to group B except that the blood was warmed (in a separate dedicated heat-exchanger unit) to 37°C. The warm blood retrograde perfusion was given continuously apart from three brief periods of 3 to 4 minutes each. In all three groups, the patient's body temperature was reduced on cardiopulmonary bypass to 32°C. A warm reperfusion (``hot shot'') consisting of 600 mL of the patient's blood containing 10 mmol/L potassium chloride was given over 3 to 5 minutes immediately before release of the cross-clamp in groups B and C.

Care was taken to monitor the perfusion pressure for all cardioplegic administrations. Coronary arterial perfusion was run at the normal diastolic pressure of the patient (70 to 80 mm Hg). Retrograde coronary sinus perfusion was run at between 25 and 50 mm Hg. Patients receiving continuous warm blood cardioplegia tended to be at the upper end of the range (40 to 50 mm Hg).

Measurement of Total Plasma Thiol Groups
Total reactive thiol groups in plasma were measured using Ellman's reagent (10 mmol/L 5,5`-dithio-bis (2-nitrobenzoic acid) dissolved in 0.1 mol/L sodium phosphate buffer, pH 7.4). The following reagents were added to new, clean plastic tubes: 50 µL of plasma, 0.55 mL of distilled water, 200 µL of sodium phosphate saline buffer, pH 7.4 (0.1 mol/L phosphate in 0.15 mol/L NaCl), and 200 µL of Ellman's reagent. Blanks were included for each sample substituting distilled water for plasma. The reaction was allowed to develop for 15 minutes at room temperature (25°C), and the resulting chromogen was measured at 412 nm spectrophotometrically. Total plasma thiols were expressed as micromoles per liter of plasma, and were calculated in nanomoles per milligram of protein, to correct for hemodilution, using a molar absorption coefficient of 13,600 L•mol<sup>-1</sup>•cm<sup>-1</sup> for the thiol-5,5`-dithio-bis (2 nitrobenzoic acid) complex. In addition, a 0.1 mol/L solution of cysteine was freshly prepared for each batch to serve as an internal standard. Samples were assayed in duplicate.

Low Molecular Mass Bleomycin-Chelatable Iron
Iron chelatable to bleomycin was determined as previously described [10]. Briefly, the reaction mixture contained DNA, bleomycin, and the plasma sample buffered to pH 7.4 with a Tris salt. In the presence of added ascorbate, iron that is able to be chelated from the plasma sample by bleomycin can degrade DNA in vitro with the release of malondialdehyde from its deoxyribose moiety, and the released malondialdehyde can be measured spectrophotometrically by reaction with 2-thiobarbituric acid.

Other Assays
Total plasma proteins were determined using a Sigma (Poole, UK) kit assay based on the Lowry technique. Plasma transferrin was quantitated by radial immunodiffusion using a polyclonal antibody to pure standards of human apotransferrin (Behring-Hoechst Ltd, Hounslow, UK).

Total plasma iron and iron-binding capacity were determined using a Sigma kit assay based on the ferrozine spectrophotometric technique. The percentage saturation of transferrin was derived from the measured total iron-binding capacity. This was found to be in close agreement with values calculated from the amount of transferrin present.

Where appropriate, values are corrected to the plasma protein content to adjust for hemodilution. All results shown are the mean of two separate assays, and statistical analyses between different time points within the groups were by analysis of variance using post tests based on Tukey-Kramer multiple comparisons. Comparisons between prebypass values and normal healthy controls were performed using Student's t test.

	Results

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Patients within the three cardioplegia groups were clinically comparable by analysis of variance for age, cross-clamp time, bypass time, and valve size (Table 1). Total plasma protein thiol levels were significantly lower (p < 0.0001) in 47 blood samples taken from patients before they underwent cardiopulmonary bypass operation (3.84 ± 0.22 nmol/mg protein) when compared with normal healthy controls (5.48 ± 0.14 nmol/mg protein), but were similar to those seen in nonsurviving adult patients with acute respiratory distress syndrome (3.56 ± 0.16 nmol/mg protein) [8]. In group A patients at the start of bypass, when cold crystalloid cardioplegia was given, a small increase in total plasma protein thiols occurred, which appeared to continue throughout the bypass procedure (Table 2). Group B patients, receiving cold blood cardioplegia, showed a greater increase in total plasma thiols when cardioplegia was initiated, and these raised values fell only slightly during the bypass procedure (see Table 2). Group C patients receiving warm blood cardioplegia showed an even greater increase in total plasma protein thiols, which was sustained throughout the bypass procedure (see Table 2). The warm blood cardioplegia value for plasma thiols after bypass was significantly greater (p < 0.05) than crystalloid cardioplegia values at the start of bypass. Plasma total protein values were also significantly higher before bypass for crystalloid and cold blood cardioplegia groups compared with bypass ``on'' samples (p = 0.05, 0.05, respectively). However, warm blood cardioplegia prebypass protein values were not significantly different than the bypass ``on'' values. There were no significant differences in protein values for any of the groups when bypass ``on'' or bypass ``off'' values were compared. Thiol values are expressed as micromoles per liter of plasma and per milligram of protein to correct for hemodilution effects. Total plasma protein values were significantly lower in all three cardioplegia groups, compared with normal healthy controls, and these values fell as a consequence of hemodilution (see Table 2). Only in the warm blood cardioplegia group did postbypass thiol values increase above the prebypass value (expressed per liter of plasma) (see Table 2). Plasma nonheme iron levels were significantly higher (0.318 ± 0.019 nmol/mg protein) in patients undergoing valve replacement operation (Table 3) compared with normal healthy controls (0.220 ± 0.02 nmol/mg protein), confirming a previous report [3]. The reason for this is at present unclear, but may represent differences in the blood sampling sites.

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The thiol group-containing molecule GSH is one of the most abundant naturally occurring thiols in living organisms and is synthesized in the reduced form. Inside the cell GSH plays a pivotal role in maintaining a reducing environment, conjugating toxins, and functioning as an antioxidant to protect against oxidative damage [reviewed in 11]. Extracellularly, however, GSH is only a minor constituent of the total plasma thiols. In blood, however, red blood cells are a major source of GSH, and have been estimated to contain 782 µmol of GSH per liter of blood. Glutathione is an important intracellular antioxidant with several key roles in protecting against oxidative stress. As a substrate for the glutathione peroxidases it can reduce hydrogen peroxide and lipid hydroperoxides, as well as maintain protein thiol groups. Extracellularly, however, the role of GSH as an antioxidant is less clear because it can also act as a prooxidant by redox cycling low molecular mass iron [12, 13]. In normal plasma the prooxidant properties of molecules with reducing properties, toward redox-active iron complexes such as GSH and ascorbate, are abolished by the iron-binding properties of transferrin and the iron-oxidizing activity of ceruloplasmin [4, 5]. The bleomycin assay for redox-active iron was introduced several years ago to detect and measure chelatable iron in plasma that was likely to take part in such prooxidant reactions. Bleomycin is not a strong enough chelator to remove iron correctly loaded onto the high-affinity iron-binding sites of transferrin, nor iron at the core of ferritin or heme [10]. Bleomycin-detectable iron usually appears in plasma when transferrin is fully saturated with iron and low molecular mass iron is available to be chelated.

Total plasma thiols have recently been shown to be remarkably good predictors of morbidity and mortality in adult patients with the acute respiratory distress syndrome [8]. Patients with abnormal left ventricular function at the time of presentation for aortic valve operation have low plasma thiol levels compared with normal healthy controls, and these values are characteristic of those seen in nonsurviving patients with acute respiratory distress syndrome. The population presenting for aortic valve operation (approximately 50 years) may well represent a group with a long-term history of exposure to oxidative stress, and this possibility is at present being considered. Patients receiving crystalloid cardioplegia did not have a significant increase in plasma thiol levels during the surgical procedures, whereas patients receiving blood cardioplegia (cold and warm) showed substantial increases in plasma thiol values (corrected for hemodilution) compared with those seen in normal healthy individuals.

Reoxygenation injury, when the heart is reperfused, is recognized as contributing to the oxidative damage received by patients undergoing cardiopulmonary bypass operation. Several studies in animals have shown changes in thiol status of reperfused hearts after periods of hypoxia, with reports of no increase in oxidized GSH [14], and increases in both oxidized and reduced GSH during early reperfusion [15]. Cross-clamp times were similar in the crystalloid and blood cardioplegia groups (see Table 1) studied, and therefore are unlikely to account for the marked difference seen in plasma thiol levels. At present the origin of increased plasma thiols seen during blood cardioplegia is unclear, but the high concentration of GSH contained within red blood cells make these a likely source to be considered. This assumption is further supported by the finding that patients receiving blood cardioplegia are more likely to show transient plasma iron overload, the iron being derived from oxidatively damaged hemoglobin.

Blood cardioplegia has been shown in animal studies to be superior to crystalloid cardioplegia in reducing oxidative damage, the protective difference being ascribed to red blood cell antioxidants such as superoxide dismutase, catalase and glutathione peroxidases [16]. Others, however, found no advantage for antioxidant protection in blood cardioplegia [17]. Supplementation of crystalloid cardioplegic solutions with a variety of free radical scavengers has indicated beneficial effects may arise in animal models from mannitol, GSH, superoxide dismutase, and a nonspecific plant peroxidase; however, evidence for specific antioxidant protection is still lacking.

Redox-active iron that is not tightly sequestered in biological systems has the potential to be cytotoxic by generating reactive and damaging oxygen intermediates. A peroxidation product of unsaturated fatty acids, namely, 4-hydroxy-2-nonenal, has been shown to be a powerful toxin and chemotactic agent. In cardiopulmonary bypass patients showing transient plasma iron overload, plasma levels of 4-hydroxy-2-nonenal are substantially increased [18].

In the present study we have monitored plasma from 47 patients receiving cold crystalloid, cold blood, and warm blood cardioplegia for changes in plasma thiol levels and iron status. The most striking finding was the increase in levels of plasma thiols, from extremely low to normal values, in patients receiving blood cardioplegia. Increased plasma thiol levels were, however, associated with a higher incidence of transient plasma iron overload, and these changes were most pronounced in the warm blood cardioplegia group.

An increase in plasma thiol levels during blood cardioplegia might be interpreted as a beneficial and protective advantage inherent in blood cardioplegia, particularly when warm blood is given. However, the higher incidence of transient iron overload, with the presence of redox active iron, after blood cardioplegia must cause some concern because GSH and protein thiols can readily react with chelatable iron to generate more reactive oxygen species [11, 12]. Our findings reinforce previous suggestions that during cardiopulmonary bypass the control of iron by chelation therapy [19] should be seriously considered, particularly when blood cardioplegia is given.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Mr Pepper and Ms Mumby thank the Wellcome Trust for their generous financial support. Doctor Gutteridge thanks the British Lung Foundation, the British Oxygen Group plc, and the British Heart Foundation for their generous support. We thank Dr Alan Heath for his advice on statistical analysis.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

1. Moat NE, Evans TE, Quinlan GJ, et al. Chelatable iron and copper can be released from extracorporeally circulated blood during cardiopulmonary bypass. Fed Eur Biochem Soc 1993;328:103–6.
2. Gutteridge JMC. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides. Fed Eur Biochem Soc Lett 1986;201:291–5.
3. Pepper JR, Mumby S, Gutteridge JMC. Transient iron-overload with bleomycin-detectable iron present during cardiopulmonary bypass surgery. Free Radic Res 1994;21:53–8.[Medline]
4. Gutteridge JMC, Quinlan GJ. Antioxidant protection against organic and inorganic oxygen radicals by normal human plasma: the important primary role for iron-binding and iron-oxidising proteins. Biochim Biophys Acta 1992;1159:248–54.[Medline]
5. Pepper JR, Mumby S, Gutteridge JMC. Sequential oxidative damage and changes in iron-binding and iron-oxidising plasma antioxidants during cardiopulmonary bypass surgery. Free Radic Res 1994;21:377–85.[Medline]
6. Voogd A, Sluiter W, Koster JF. The increased susceptibility to hydrogen peroxide of the (post) ischemic rat heart is associated with the magnitude of the low molecular weight iron pool. Free Radic Biol Med 1994;16:453–8.[Medline]
7. Flaherty JT, Weisfeldt ML. Reperfusion injury. Free Radic Biol Med 1988;5:409–19.[Medline]
8. Quinlan GJ, Evans TW, Gutteridge JMC. Oxidative damage to plasma proteins in adult respiratory distress syndrome. Free Radic Res 1994;20:289–98.[Medline]
9. Quinlan GJ, Evans TW, Gutteridge JMC. 4-Hydroxy-2-nonenal levels increase in the plasma of patients with adult respiratory distress syndrome as linoleic acid appears to fall. Free Radic Res 1994;21:95–106.[Medline]
10. Gutteridge JMC, Hon Y. Iron complexes and their reactivity in the bleomycin assay for radical-promoting loosely-bound iron. Free Radic Res Commun 1986;2:143–51.[Medline]
11. Meister A. On the antioxidant effects of ascorbic acid and glutathione. Biochem Pharmacol 1992;44:1905–15.[Medline]
12. Tien M, Bucher JR, Aust SD. Thiol-dependent lipid peroxidation. Biochem Biophys Res Commun 1982;107:279–85.[Medline]
13. Gutteridge JMC. Reduction of low molecular mass iron by reducing molecules present in plasma and the protective action of caeruloplasmin. J Trace Elem Electrolytes Health Dis 1991;5:279–81.[Medline]
14. Darley-Usmar VM, O'Leary V, Stone D. The glutathione status of perfused rat hearts subjected to hypoxia and reoxygenation: the oxygen paradox. Free Radic Res Commun 1989;5:283–9.
15. Lesnefsky EJ, Repine JE, Horwitz LD. Oxidation and release of glutathione from myocardium during early reperfusion. Free Radic Biol Med 1989;7:31–5.[Medline]
16. Julia PL, Buckberg GD, Acar C, et al. Studies of controlled reperfusion after ischemia. XXI. Reperfusate composition: superiority of blood cardioplegia over crystalloid cardioplegia in limiting reperfusion damage-importance of endogenous oxygen free radical scavengers in red blood cells. J Thorac Cardiovasc Surg 1991;101:303–13.[Abstract]
17. Bical O, Gerhardt M-F, Paumier D, et al. Comparison of different types of cardioplegia and reperfusion on myocardial metabolism and free radical activity. Circulation 1991;84(Suppl 3):375–9.
18. Quinlan GJ, Mumby S, Pepper JR, Gutteridge JMC. Plasma 4-hydroxy-2-nonenal levels during cardiopulmonary bypass, and their relationship to the iron-binding of transferrin. Biochem Mol Biol Int 1994;34:1277–82.[Medline]
19. Menasché P, Antebi H, Alcindor L-G, et al. Iron chelation by deferoxamine inhibits lipid peroxidation during cardiopulmonary bypass in humans. Circulation 1990;82(Suppl 4):390–6.

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