Cardiac surgery: myocardial energy balance, antioxidant status and endothelial function after ischemia–reperfusion

Biomed Pharmacother 56 (2002) 483–491 www.elsevier.com/locate/biopha

Dossier: Free amino acids in human health and pathologies

Cardiac surgery: myocardial energy balance, antioxidant status and endothelial function after ischemia–reperfusion F. Carlucci a,*, A. Tabucchi a, B. Biagioli b, F. Simeone b, S. Scolletta b, F. Rosi a, E. Marinello a a

Institute of Biochemistry and Enzymology, University of Siena, Nuovi Istituti Biologici, Via Aldo Moro, 53100 Siena, Italy b Institute of Thoracic and Cardiovascular Surgery and Biomedical Technologies, University of Siena, Siena, Italy

Abstract Myocardial and endothelial damage is still a widely debated problem during the ischemia–reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Ischemia; Oxidative stress; Purine metabolism

1. Introduction Organs undergoing ischemia–reperfusion in vivo are extensively damaged [19]. This is a critical problem associated with procedures such as coronary artery by-pass graft (CABG), being linked to a series of events such as block of oxidative phosphorylation, nitric oxide and free radical production [32] and endothelial dysfunction. All these alterations have been extensively characterized under experimental conditions [10], but there have been few data on their relationships at clinical level. Improved in vivo and ex * Corresponding author. E-mail address: [email protected] (F. Carlucci). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 7 5 3 - 3 3 2 2 ( 0 2 ) 0 0 2 8 6 - X

vivo myocardial protection is crucial for the successful therapy of patients with end-stage cardiac dysfunction, but despite much research effort no single therapy has been adopted clinically for protection of the ischemic/reperfused myocardium, a goal that is commonly referred to as “cardioprotection”. The primary aim of this study was to investigate the effects of CABG on high energy phosphates and oxidative and antioxidant metabolism at cellular level. We also studied plasma parameters that reflect free radical production and subsequent endothelial damage, related to the ischemia–reperfusion sequence in heart surgery. This was done in myocardial biopsies obtained before heart cardioplegic arrest, at the end of ischemia time and

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30 min after reperfusion. Purine nucleotides, nucleosides and bases, NAD and glutathione in its reduced and oxidized forms (GSH, GSSG) were evaluated in an extract of biopsy material. At the same times we obtained blood samples in which we evaluated nitric oxide (measuring its stable end-products, nitrate and nitrite), plasma glutathione peroxidase (plGPx), endothelin-1 (ET-1) and reactive oxygen metabolites (ROM). Nucleotide and glutathione determinations are widely accepted measures of cellular energy, antioxidant status and organ preservation [38,41]. The role of purine compounds in the matter and energy metabolism is well known [26]. ATP, ADP and AMP are in equilibrium in cells and have definite concentrations, from which it is possible to evaluate cell energy charge (EC), phosphorylation potential (ATP/ADP) and integrity. Nucleosides and free purine bases are products of nucleotide catabolism and provide information about total regulation of purine metabolism. The nucleotide/(nucleoside + base) ratio is an index of the balance of purine anabolic reaction over catabolic reactions [11,12]. Among adenine nucleotides, the oxidized form of nicotinic coenzyme (NAD), is useful for evaluating redox potential, and as an indicator of post-ischemic heart function [33]. NAD also plays a central role in the glutathione redox cycle and in poly(ADP-ribose) polymerase activity [5]. Glutathione has oxidized (GSSG) and reduced (GSH) forms, which reflect redox state and cell defenses against oxygen toxicity. The GSH/GSSG ratio is an index of cell redox modification like other well known compounds such as vitamin E and coenzyme Q. Glutathione is currently evaluated during oxidative stress and is regarded as a major antioxidant [9]. Antioxidant status is modified almost exclusively through the mediation of highly reactive molecules, the so-called “free radicals”, which are ROM including superoxide and hydroxyl free radicals and hydrogen peroxide. Cell antioxidant capacity and oxidant status are the best markers of cell condition. Endothelial injury is another aspect of ischemia and reperfusion damage that causes significant morbidity and mortality in cardiovascular diseases. Endothelial cell death may contribute to the hypoxic and reperfusion phase of CABG. The mechanisms of hypoxic endothelial cell death are not known but may involve calcium influx, mitochondrial dysfunction and purine nucleotide depletion [1,24,43]. Recent studies have demonstrated that release of nitric oxide (NO) from coronary artery endothelium decreases after myocardial ischemia and reperfusion, by an impaired response to endothelium-dependent vasodilators [16]. Decreased NO release by the coronary vascular endothelium may be an important component in the pathogenesis of myocardial ischemia–reperfusion injury.

ET-1 is a major endothelium-derived factor and is the most potent vasoconstrictor known. A number of recent studies have evaluated the behavior of this peptide in relation to the ischemia–reperfusion sequence, free radical production and apoptosis [28,45].

2. Materials and methods 2.1. Patients We studied 14 patients (age 47–70 years, mean 64.5) undergoing coronary artery by-pass surgery for ischemic cardiopathy. Six had plurivasal and eight monovasal cardiopathy. The patients were classified as level 2 of the clinical severity score of Higgins et al. [23]. 2.2. Surgical techniques Antiangina medication (nitrates and calcium-blockers) was continued until the morning of the operation. Anesthesia was induced with fentanyl (5 µg/kg) and diazepam (0.3 mg/kg) and maintained with fentanyl infusion (25 µg/kg per min). Muscle relaxation was achieved with pancuronium bromide (0.1 mg/kg). Ventilation with a mixture of oxygen and nitrous oxide (50:50) was adjusted to maintain normocapnia. During by-pass hematocrit was maintained between 20 and 25% and pump flows were maintained between 2.0 and 2.2 l/min per ml2. Mean arterial pressure was maintained between 60 and 70 mmHg with sodium nitroprusside (0.5–5 µg/kg per min) or noropinephrine (0.01–0.5 µg/kg per min). 2.3. Cardioplegic techniques Cold blood cardioplegia was performed with a 4:1 ratio of oxygenated circuit blood and crystalloid solution. The physical and biochemical composition of the blood cardioplegic solution was according to the protocol of the UCLA Medical Center [7] except for the absence of L-aspartate and L-glutamate. Delivery was divided between antegrade and retrograde through the aorta and coronary sinus. Diastolic arrest was achieved with an initial cold (4–8 °C) blood high-potassium solution (20–25 mEq/l) at a flow rate of 200–300 ml/min for 2 min through the aorta and 2 min through the coronary sinus. Coronary sinus perfusion pressure was kept below 40 mmHg. On completion of each distal–proximal anatomosis or at 20 min intervals, a flow rate of 150–200 ml/min potassium solution (8–10 mEq/l) was administered for 1 min through the aorta and for 1 min through the coronary sinus. Before cross-clamp release warm (37 °C) blood low-potassium solution was infused at 150 ml/min and

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50 mm Hg for 3–5 min, alternating between aortic root and coronary sinus (modified cardioplegic reperfusion according to Buckberg) [8]. Moderate systemic hypothermia (28–30 °C) and topical hypothermia were used.

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The background electrolyte was borate buffer (20 mM). The conditions were pH 10.00, 20 kV and 12 s hydrostatic load at 26–27 °C. The electric field was 333.33 V/cm with a current of approximately 80 µA, according to Carlucci et al. [12].

2.4. Myocardial biopsies and blood samples 2.6. Plasma parameters Transmural left ventricular biopsy specimens, obtained with Tru-cut biopsy needles, were immediately washed of blood in ice-cold, isotonic buffer solution and frozen in liquid nitrogen (within 20 s). Samples were obtained immediately before aortic cross-clamping (t1), immediately before clamp removal (t2) and 30 min after reperfusion (t3), from the same area at each time for all patients: they were homogenized at 10% with 0.4 N perchloric acid (PCA) in 0.5 ml tubes using a nylon motor pestle. For practical handling of the extract, not less than 20 µl were used for biopsy specimens under 2 mg wet-weight. Extracts were then centrifuged (12 000 × g for 10 min) in a cooled microfuge. The pellet was resuspended in 0.1 M NaOH using the same volume as for PCA extraction. Protein content of the resuspended pellet was determined by the Coomassie brilliant blue binding procedure described by Bradford [6] with Bio-Rad protein reagent, using crystalline bovine serum albumin as standard. The supernatants were neutralized with 2.7 M potassium hydroxide. Correct neutralization is critical in such small sample volumes. Neutralized samples are stable for several weeks. Potassium perchlorate was removed by freeze–thawing with a subsequent centrifugation at 12 000 × g for 3 min. Aliquots of the extracts were analyzed by CE. Our CE procedure made it possible to carry out the analysis of purine compounds with a very small quantity (0.6 mg) of tissue. The energy charge of adenylates and the nucleotide/(nucleoside + base) ratio were calculated as follows:

Plasma ET-1 was determined by radioimmunoassay kit (Amersham, UK). Plasma glutathione peroxidase was determined using hydrogen peroxide as substrate and immunoreactivity determined by ELISA (CalbioChem, USA). ROM were determined by colorimetric assay (Diacron ITA) and are expressed as Carratelli units (Ucarr) [17]. One UCarr is equivalent to a hydrogen peroxide concentration of 0.08 mg%. Direct measurement of NO in biological samples is problematical, as its half life is only 10–30 s. The more stable end-products of NO metabolism, nitrate and nitrite, are usually measured instead [15]. Nitrate and nitrite were measured by a capillary electrophoresis method according to Davies et al. [15]. Plasma samples were ultrafiltered with 5K cartridges to remove protein excess. Separation conditions were: 40 cm × 50 mm i.d. uncoated capillary; –10 kV voltage; 150 mM NaCl/5 mM Tris–HCl buffer containing 2 mM TTAB (pH 7.4). Total analysis time 5 min. Analyte values were corrected for blood dilution (oxygenated circuit blood and crystalloid solution) on the basis of hematocrit variations. 2.7. Statistical analysis Differences between the various times were analyzed by the Kruskal–Wallis test. Correlation analysis was performed with Spearman’s test. Significant differences were assumed for p < 0.05 and indicated in tables and figures.

EC = (½ADP + ATP)/(AMP + ADP + ATP) Nt/(Ns + B) = (AMP + ADP + ATP + GMP + GDP + GTP + IMP)/(Ado + Ino + Hx) Blood samples were obtained from the coronary sinus at the same time as the myocardial biopsies (t1, t2, t3); they were immediately centrifuged at 2000 × g and the plasma frozen in liquid nitrogen. 2.5. CE procedure for cellular parameters A Waters Quanta 4000 instrument (Waters Chromatography Division, Milford, MA, USA) was used for all electrophoretic separations. Analysis was performed with a Supelco bare CEIect-Fs column (Supelco Inc., Bellafonte, PA, USA) (60 cm × 75 µm i.d.) with the window at a distance of 52.5 cm. The results were read at 185 nm.

3. Results Data on purine metabolites are referred to protein content that showed no significant change between subjects or over the experimental time-course (data not shown); thus excluding variation in tissue water content. The basal value of energy charge ATP/ADP ratio, GSH/GSSG ratio and nucleotide/(nucleoside + nucleobase) ratios were 0.72 ± 0.08; 1.68 ± 0.15; 10.14 ± 1.68 and 10.56 ± 2.51, respectively. Plasma values of nitrate/nitrite, ET-1, plGPx and ROM were 30.18 ± 5.58 µM, 3.83 ± 0.98 fmol/ml, 23.61 ± 9.22 µg/ml and 287.56 ± 55.85 Ucarr, respectively. During the ischemic period (t2) we observed a significant decrease in ATP and GTP levels and an increase in their catabolic products AMP, GMP, IMP, adenosine, inosine and

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hypoxanthine; no significant accumulation of diphosphates (ADP, GDP) was evident. The energy charge, ATP/ADP and nucleotide/(nucleoside + nucleobase) ratios decreased significantly. No appreciable xanthine and uric acid levels were evident, confirming low activity of human myocardial xanthine oxidoreductase [36]. Ischemia also induced a decrease in myocardial levels of nicotinic coenzyme (NAD) from 4.79 ± 1.23 nmol/mg protein to 3.31 ± 0.93 nmol/mg protein (p < 0.05). Nicotinic coenzyme (NAD) levels were reduced, a consequence of the blocking of NADH oxidation in the respiratory chain, determined by reduced availability of molecular oxygen, last electron acceptor in the integral membrane electron transport pathway. Reduced glutathione levels showed a sharp reduction. GSH is probably oxidized to GSSG as organic hydroperoxide and hydrogen peroxide are reduced during ischemia. IMP levels rose up to 89% of basal value during the ischemic phase. The GSH/GSSG ratio decreased to 30% of basal value. Table 1 shows nucleotide, nucleoside and base values normalized for protein content in myocardial tissue at the various times. Table 2 reports the content of oxidized nicotinic coenzyme and oxidized and reduced glutathione. Fig. 1 shows the principal ratios calculated from metabolite content. Variations in ATP levels between basal and postischemic phase (∆t1 – t2) showed a positive correlation with those of NAD at the same times (Fig. 2) (p < 0.05). At the end of the ischemic phase (t2), we observed a significant reduction (about 50%) in nitrate plus nitrite concentrations, in plasma from the coronary sinus. Glutathione peroxidase levels decreased from 23.6104 ± 4.28 µg/ml (t1) to 15.04 ± 4.16 µg/ml (t2) (p < 0.05), while ROM and ET-1 did not change (Fig. 3). After 30 min of reperfusion (t3) concentrations of monophosphate nucleotides (AMP, GMP, IMP) returned to basal values. Nucleoside and base levels remained significantly higher, though a decrease was recorded. The energy charge did not vary with respect to t2. The ATP/ADP ratio showed

Fig. 1. Mean energy charge, ATP/ADP ratio, nucleotides/(nucleosides + nucleobases) ratio and GSH/GSSG ratio. Bars indicate S.D. * P < 0.05 with respect to values at t1. Table 2 Myocardial antioxidant status during coronary artery by-pass surgery. Values are means (nmol/mg protein) ± S.D. n = 14

t1

t2

t3

GSH GSSG NAD .

7.51 ± 1.26 0.77 ± 0.16 4.79 ± 1.23

3.88 ± 0.22* 1.76 ± 1.11* 3.31 ± 0.93*

3.12 ± 0.73* 2.61 ± 0.30* 3.82 ± 1.77*

* P ≤ 0.05 with respect to t1.

Table 1 Myocardial purine nucleotide content during coronary artery by-pass surgery. Values are means (nmol/mg protein) ± standard deviation (S.D.) n = 14

t1

t2

t3

Ado Ino Hyp IMP AMP ADP ATP GMP GDP GTP .

0.88 ± 0.21 1.50 ± 0.42 1.19 ± 0.48 2.11 ± 0.52 5.03 ± 1.25 11.16 ± 2.59 15.84 ± 2.33 0.32 ± 0.18 1.15 ± 0.28 1.94 ± 0.24

2.71 ± 0.32* 2.98 ± 0.87* 3.20 ± 0.73* 3.98 ± 0.69* 8.68 ± 1.29* 13.56 ± 2.76 9.3 ± 3.47* 0.94 ± 0.22* 1.77 ± 0.29 0.38 ± 0.09*

1.70 ± 0.35* 2.69 ± 0.74* 1.82 ± 0.27* 3.02 ± 0.50 7.56 ± 1.12 12.78 ± 2.13 10.05 ± 2.63* 0.51 ± 0.16 1.69 ± 0.44 0.84 ± 0.17*

* P ≤ 0.05 with respect to t1.

Fig. 2. Correlation between the variation of ATP and NAD levels. r = 0.6879 according to Spearman’s test. P < 0.01.

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Fig. 3. Behavior of plasma parameters. Bars indicate S.D. * P < 0.05 with respect to values at t1.

an increasing trend with respect to t2 but was significantly lower than at t1. The nucleotide/(nucleoside + nucleobase) ratio recovered to some extent due to a concomitant reduction in nucleoside and base levels and to an increase in high energy phosphates (Fig. 2). In the evaluation of the antioxidant status at t3, we observed a further decrease in cellular GSH/GSSG ratio (Fig. 2); this decrease (40%) was less evident than the variation recorded showed between t1 and t2 (75%), indicating different rates of oxidative insult. Plasma concentrations of nitrate/nitrite increased to 21.83 ± 5.23 µM with respect to t2, but were still significantly lower than at t1. ET-1 was significantly enhanced (5.64 ± 0.84 fmol/ml) with respect to t1 (3.43 ± 1.06 fmol/ml). Glutathione peroxidase and ROM were essentially unchanged with respect to t1 (Fig. 3).

4. Discussion In CABG, the heart undergoes a three-step sequence of events (arrest, ischemia and reperfusion), during which

myocyte damage occurs due to ATP break-down and depletion of the naturally occurring defense mechanisms against free radical injury. In this situation, endothelial function is also impaired contributing to the development of organ dysfunction. Extracorporeal circulation could be another potential source of oxidative stress. 4.1. Purine nucleotide metabolism When generation of ATP by adenosine diphosphate rephosphorylation is inhibited by the lack of available oxygen, ADP is converted into adenosine triphosphate and adenosine monophosphate by myokinase and does not accumulate. AMP is then broken down to purines via two pathways. In the AMP→IMP→inosine pathway, AMP deamination to IMP by AMP deaminase precedes dephosphorylation of IMP to inosine by 5'-nucleotidase. In the AMP→adenosine pathway, AMP itself is dephosphorylated by 5'-nucleotidase [50]. The accumulation of adenosine and IMP detected in this study shows that both these catabolic pathways play major roles in the human myocardium. IMP production exceeds its role as catabolic product, being an

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AMP-S

Krebs cycle

7 2

Aspartate GDP, Pi

1

Fumarate GTP ATP

5

2 ADP

AMP

IMP

ratio with respect to t2. Monophosphate nucleotides returned to basal levels, high-energy phosphate levels increased slightly and nucleosides and bases tended to decrease. The energy charge and the ATP/ADP ratio remained low. These results indicate that oxidative metabolism, impaired during ischemia, was partially reversed after reperfusion, but release of purine catabolites persisted for longer than 30 min, in line with previous reports [39].

4 3 5

6

4.2. Oxidative stress and antioxidant system

Inosine NH3

1. Adenylosuccinate syntherase 3. AMP deaminase 5. 5'-nucleotidase 7. Glutamate oxalacetate transaminase

Adenosine

2. Adenylosuccinate lyase 4. Adenylate kinase 6. Adenosine kinase

Fig. 4. Purine nucleotide cycle.

intermediate in the purine nucleotide cycle (Fig. 4), which has a role in the recovery of energy state during reperfusion [2]. It has been demonstrated that the major metabolic event during ischemia is a temporarily saving of the nucleotide pool as inosine-5'-monophosphate (IMP). When circulation resumes and energy state recovers, IMP is in fact converted back to AMP via the purine nucleotide cycle [25] and its basal level tends to be restored (Table 1). It has also been demonstrated that IMP produced in organs suffering ischemia–reperfusion injury exerts a broad spectrum of activity in the down-regulation of neutrophil recruitment, and can be considered a cardioprotective agent [35]. During the ischemic period (t2), the reduction in ATP and GTP levels was not reflected by an accumulation of ADP and GDP, which were rapidly converted to AMP, GMP and IMP, and then to nucleosides and bases, the principal catabolites responsible for a deterioration in energy charge. The decreased nucleotide/(nucleoside + base) ratio reflects the prevalence of myocardial purine catabolic reactions in the ischemic phase. High adenosine levels (three-fold those at t1) indicated a major increment in the activity of the 5'-nucleotidase pathway. The reduction in NAD levels during the ischemic phase is probably related to blocking of NADH oxidation in the respiratory chain, determined by reduced availability of molecular oxygen, which is the last electron acceptor in the integral membrane electron transport pathway. Depletion of NAD and ATP is related to polyADPribose polymerase (PARP) activation after myocardial ischemia as previously reported [34]. The positive correlation between the charge in ATP and NAD concentrations in the interval t1–t2 reflects a close relationship of these two nucleotides during the ischemic period. After reperfusion (t3), there was an inversion of the catabolic trend, as shown by an increase in the Nt/(Ns + B)

Oxygen-derived free radicals are important agents of tissue injury during ischemia and reperfusion. Variations in ROM levels were not evident during the ischemic phase nor during reperfusion (Fig. 3). The short half-life of these molecules is well known [29] and they only seem to be generated in the early stage of reperfusion [22,42]. The time of evaluation (30 min after reperfusion) used here could be too late to detect them. We found an imbalance in the cellular GSH/GSSG ratio, plasma glutathione peroxidase activity and plasma concentrations of nitrates/nitrites (i.e. NO release from coronary artery) during ischemic phase and reperfusion, which is indirect evidence of free radical production before organ reoxygenation. Oxidant injury and stunning in cardiac tissue during ischemia before reperfusion [3,4] and its association with increased cell death up to reperfusion have been reported [31]. The behavior of plasma GPx was not associated with the cellular glutathione system, since it decreased during the ischemic phase but remained unchanged during reperfusion. Impairment of GPx expression has, however, been reported in relation to oxidative stress [14] and in a different situation cellular and plasma antioxidant systems have been observed to act in opposite ways [49]. An alteration in plasma GPx expression related to cardiopulmonary by-pass cannot be excluded as the procedure implicates systemic inflammatory response and cytokine release [13]. It has recently been demonstrated that extracorporeal circulation in dialysis patients increases the risk of elevated free radical production [18]. 4.3. Endothelial dysfunction The role of endothelial-derived nitric oxide in ischemia– reperfusion injury is still debated. Several experimental studies have found that it exerts a cardioprotective effect during myocardial ischemia–reperfusion. Conversely, other studies suggest that NO exacerbates reperfusion injury by inducing the production of peroxynitrite. Nevertheless, inducible and constitutional myocardial nitric oxide synthase (NOS) isoenzymes are regarded to be inversely regulated after ischemia–reperfusion in experimental and

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clinical studies, with a general down-regulation of constitutive NOS and up-regulation of inducible NOS [47]. We showed a significant decrease of nitrate plus nitrite levels during both ischemic and reperfusion phases. Free radical production and antioxidant cell system imbalance during the ischemic phase (t2) could lead to alteration of NOS and/or to modulation of NO bioactivity [27]. At experimental level, it is well established that ischemia–reperfusion injury is related to diminished endothelial release of NO with a concomitant oxygen free radical burst [44]. During CABG surgery, other causes may concur to realize it: (1) L-arginine depletion during cardioplegic arrest; (2) endothelial dysfunction induced by hyperkalemic solutions; (3) depressed activity of NOS synthase due to low myocardial temperature [20]. After reperfusion with blood (possibly containing L-arginine), NO release continued to decrease, suggesting real endothelial dysfunction in line with other reports [37]. Reduced NO release by the coronary vascular endothelium may be an important component in the pathogenesis of myocardial injury during CABG [30]. A recent study reports that systemic and myocardial lipid peroxidation is lower in off pump coronary artery by-pass grafting than in CABG, shifting the problem to the role of extracorporeal circulation [46]. Upon reperfusion, we observed significant enhancement of ET-1 levels, which could be related to free radical injury and/or to its relationship with NO production. As previously shown, free-radical-catalyzed lipid peroxidation stimulates ET-1 expression on endothelial cells via isoprostane production [48] and may play a role in myocardial ischemia/reperfusion injury [16]. Furthermore, NO inhibits ET-1 production by blood vessels [21]; thus when NO activity is reduced after ischemia–reperfusion, its inhibition of ET-1 production is attenuated, which may lead to enhancement of ET release from the vasculature, as reported in the present study.

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of the normal energy balance for up to 30 min after reperfusion. It is worth recalling, however, that the results of CABG are by now excellent, especially for low risk patients and any causal relationship between high-energy phosphate and mechanical function is still debated. On the other hand, it has been reported that ATP content at the end of ischemia is also an accurate predictor of potential ATP resynthesis during reperfusion and that the recovery of myocardial function is achieved on recovery of high-energy phosphate [40] showing the critical importance of maintaining highenergy phosphate at the highest possible level during ischemic arrest. In addition, our research shows that adenosine is not the main compound derived from ATP degradation in the human heart, and that the AMP deaminase pathway leading to IMP production is also relevant. During aortic crossclamping, an imbalance of purine metabolism occurs. We showed that in the prolonged ischemic period necessary for coronary artery by-pass surgery IMP is a key metabolite and that nucleotide catabolism may increase through the contribution of the 5'-nucleotidase pathway. Attempts to maintain adequate levels of purine compounds during cardiac ischemia should consider ATP degradation in its entirety. The metabolic pathways of purines are possible targets for drugs in cardioplegic solutions for routine heart surgery and heart transplants. In the last decade, experimental data on ischemia–reperfusion injury has greatly increased, enabling improvement of myocardial preservation techniques. Nevertheless, an in vivo overall interpretation of this complex phenomenon is still mainly speculative. For such a view, our data could provide new insights into myocardial and endothelial behavior during surgical procedures and could be useful for developing new cardioprotective strategies, since continuous refinements in the practice of myocardial protection are necessary to optimize post-surgical cardiac function.

References 5. Conclusion CABG implicates various changes in myocardial energy balance, antioxidant status and endothelial function. The contribution of cardioplegic-induced arrest and cardiopulmonary by-pass (i.e. reduction of nitrate/nitrite and plasma glutathione peroxidase during the ischemic phase) could exacerbate ischemic damage, leading to suboptimal recovery of cardiac function after reperfusion. We have no direct evidence of ROM production, however, our data on cellular GSH, plasma nitrate/nitrite and plasma ET-1 clearly indicate that oxidative stress is an important feature during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration

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