Eur J Cardiothorac Surg 2006;30:41-48
© 2006 Elsevier Science NL
Changes in myocardial free amino acids during pediatric cardiac surgery: a randomised controlled trial of three cardioplegic techniques
Paul Modi,
Saadeh M. Suleiman,
Barnaby C. Reeves,
Ash Pawade,
Andrew J. Parry,
Gianni D. Angelini,
Massimo Caputo
*
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK
Received 14 November 2005;
received in revised form 8 March 2006;
accepted 10 March 2006.
* Corresponding author. Tel.: +44 117 928 2979; fax: +44 117 929 9737. (Email: m.caputo{at}bristol.ac.uk).
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Abstract
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Objective: The developing heart has a much greater dependence on amino acid (AA) metabolism than the adult heart in determining its ischemic tolerance. Blood cardioplegia preserves myocardial free AAs in adult hearts but no clinical studies have looked at the effect of different cardioplegic techniques on intracellular free AAs in the pediatric heart. Methods: Pediatric patients were randomised to receive intermittent antegrade cold crystalloid (CC), cold blood (CB) or cold blood cardioplegia with a ‘hot shot’ (CB + HS). Right ventricular biopsies were collected prior to ischemia, at the end of ischemia and 20 min after reperfusion. Amino acid levels were analysed as repeated measures, adjusting for baseline levels. Data were analysed separately for acyanotic and cyanotic patients. Results: Of 103 patients recruited, 32 (22 acyanotic and 10 cyanotic), 36 (24/12) and 35 (25/10), respectively were allocated to CC, CB and CB + HS groups. Cyanotic patients were significantly younger with longer cross-clamp times. In acyanotic patients, there were no significant effects of cardioplegic method on aspartate, glutamine, taurine, alanine or branched chain AA levels (all p
> 0.05). However, in cyanotic patients, there were significant interactions of cardioplegic method and time (all p
< 0.05) for all amino acids, with patients allocated to CB + HS having higher levels after reperfusion compared with CC, and patients allocated to CB having intermediate levels. Conclusions: For cyanotic patients (younger, longer cross-clamp times), CB + HS preserves myocardial free AAs better than CC; CB gives an intermediate effect. In acyanotic patients, AA levels (all p
> 0.15) and group means were similar both at the end of ischemia and after reperfusion.
Key Words: Cardioplegia • CHD • Cyanotic • CHD • Acyanotic • Ischemia/reperfusion • Pediatric • Surgery
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1. Introduction
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Although a substantial amount of data is available concerning the beneficial effects of cold blood cardioplegia (CB) and terminal warm blood cardioplegic reperfusion (‘hot shot’, HS) compared with cold crystalloid cardioplegia (CC) in the adult myocardium, much less is known about these in the developing heart. It would be unwise to uncritically extrapolate cardioprotective strategies originally developed in adult hearts to the pediatric setting due to structural, functional and metabolic differences between these two groups [1]. Rather, approaches to pediatric myocardial protection must be studied in the developing heart, ideally in the clinical setting in order to avoid the effects of species-related differences that are evident in animal models [2]. Studies must also take account of cyanosis which is often present in young hearts undergoing cardiac surgery and which alters not only the sensitivity of the heart to ischemia-reperfusion but also its responsiveness to cardioplegia [3].
In a randomised clinical trial, we recently observed that cold blood cardioplegia with a hot shot (CB + HS) reduces the fall in ATP compared with cold crystalloid cardioplegia (CC) in cyanotic but not acyanotic children [4]; CB on its own had intermediate effects. However, adenine nucleotides are only one piece of the jigsaw; amino acids (AAs) such as glutamate and aspartate play an important role as intermediate metabolites in the ischemic myocardium where they can be used as energy substrate under anaerobic conditions. In adults, CB preserves myocardial free AAs as well as adenine nucleotides to a greater extent than CC [5,6]. Amino acid metabolism is much more important in the immature myocardium which derives its greater ischemic tolerance from AA transamination and substrate level phosphorylation [7]. However, no clinical studies have looked at the effect of different cardioplegic techniques on free AAs in the pediatric heart. The elucidation of changes in the concentrations of AAs which are important for maintaining normal cardiac cellular function should help to formulate cardioprotective strategies that lead to better preservation of these metabolites in immature hearts during cardiac surgery.
Our aim was to compare the effects of three cardioplegic techniques (CC, CB and CB + HS) on the myocardial concentrations of amino acids in cyanotic and acyanotic pediatric patients undergoing cardiac surgery. The results of adenine nucleotide concentrations in the same trial have been recently published [4].
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2. Methods
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Pediatric patients undergoing elective repair of congenital heart defects over an 18-month period at the Royal Hospital for Children, Bristol, were randomised to:- 1. CC (St. Thomas’ I crystalloid cardioplegia, 20 mM KCl, 16 mM MgCl2, 2.2 mM CaCl2, 144 mM NaCl, 1.0 mM procaine HCl), or
- 2. CB (4:1 dilution blood/St. Thomas’ I crystalloid cardioplegia to give the same at-patient concentrations), or
- 3. CB + HS (HS identical to CB except at 37 °C).
Random allocations to groups were printed on cards concealed inside sealed, numbered, opaque envelopes. The next numbered envelope was opened at the time of surgery, after a patient had been definitively recruited. The study was approved by the local Institutional Review Board and a written parental consent was obtained in all cases.
2.1 Anesthetic and surgical technique
Anesthetic technique was standardized as reported previously [8]. Cardiopulmonary bypass (CPB) was established between ascending aortic and bicaval cannulae with moderate systemic hypothermia (28–32 °C). After the aorta was cross-clamped, either CC or CB was infused into the aortic root at 4 °C. The induction dose was 110 ml/(m2
min) antegradely for 4 min with a maintenance dose of 110 ml/(m2
min) for 2 min at 20–30 min intervals. Aortic root pressure was maintained between 40 and 50 mmHg during cardioplegic delivery and topical cooling with cold saline solution (4–6 °C) was used in all patients. The ‘hot shot’ was identical in composition to the induction and maintenance doses of blood cardioplegia and was administered at 110 ml/m2/min for 2 min at 37 °C immediately prior to unclamping the aorta.
2.2 Collection of ventricular biopsies for amino acid analysis
Myocardial biopsies (mean weight ± SD, 3.6 ± 2.2 mg) were collected from the anterior wall of the right ventricle (inlet) using an 18 G x 6 cm Trucut biopsy needle (Allegiance Healthcare, IL, USA) immediately prior to cross-clamping the aorta (control biopsy), just before releasing the cross-clamp (ischemic biopsy) and after 20 min reperfusion (reperfusion biopsy). Each specimen was immediately frozen in liquid nitrogen until processing for the analysis of AAs by high-performance liquid chromatography (Waters HPLC system). Details of sample preparation and analysis have been described previously [9].
2.3 Statistical analysis
Amino acid levels are summarised using geometric means with 95% confidence intervals (CI). Metabolic data are presented graphically as geometric mean and standard errors. Because of the small sample size, missing data (6%, never missing for both time points) were interpolated on the basis of the data for other patients in the same group. A natural logarithmic transformation was applied to the data for all variables to normalise their distributions. Data were analysed with mixed models (SAS version 8), fitting the between-subject factor of ‘type of cardioplegia’ and the within-subject factor of ‘time’ (repeated measurements over time), and the interactions of these two factors; separate analyses were carried out for acyanotic and cyanotic children. Overall statistical tests of the equality of the geometric means for the three groups were carried out for both post-operative time points, i.e. at the end of ischemia and 20 min after reperfusion. The baseline level (obtained from the first biopsy) was entered as a covariate.
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3. Results
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A total of 103 children were recruited to the study (71 acyanotic and 32 cyanotic). Twenty-two, 24 and 25 acyanotic children were randomly allocated to CC, CB and CB + HS cardioplegia, respectively; 10, 12 and 10 cyanotic children were randomly allocated to the corresponding groups. There were no complications of biopsy collection. Cyanotic patients tended to be younger and had longer CPB and aortic cross-clamp times (Table 1
). The diagnoses for these patients are shown in Table 2
. The effects of the three cardioplegic techniques on amino acid levels were dramatically different in cyanotic and acyanotic children, i.e. there were interactions of cyanosis and type of cardioplegia. Therefore, although the trial was not originally conceived in this way, we report the results separately for cyanotic and acyanotic subgroups. The characteristics of patients, and baseline amino acid levels, were similar in the three arms of the cyanotic and acyanotic groups (see Tables 1, 3 and 4
).
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Table 3. Geometric means and confidence intervals for amino acid levels at the end of ischemia and 20 min after reperfusion in cyanotic children, by cardioplegia group
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Table 4. Geometric means and confidence intervals for amino acid levels at the end of ischemia and 20 min after reperfusion in acyanotic children, by cardioplegia group
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3.1 Cyanotic subgroup
There were marked interactions of cardioplegic technique and time for most of the seven amino acids investigated. These interactions are shown in Fig. 1
and are described quantitatively in Table 3, in which we report separate statistical tests of the overall effect of cardioplegic technique: (a) at the end of ischemia and (b) 20 min after reperfusion. For most amino acids (aspartate, alanine, glutamine, taurine), there were no differences in amino acid levels between groups at the end of ischemia (p ranging from 0.23 to 0.72) but marked differences 20 min after reperfusion (p ranging from <0.0001 to 0.007).
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Fig. 1. Geometric means and standard errors for aspartate (A), alanine (B), branched-chain amino acids (C), glutamine (D), taurine (E) and alanine/glutamate ratio (F) in cyanotic children at the end of ischemia and 20 min after reperfusion.
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These effects can be seen in the graphs as a ‘fanning’ out of the lines for each group from a similar level at the end of ischemia to different levels 20 min after reperfusion. Except for branched-chain amino acids (BCAA, p
= 0.09) and the alanine/glutamate ratio (a marker of ischemic stress, p
= 0.45), the means for the CB + HS group 20 min after reperfusion were significantly higher than for the CC group (p
< 0.005). The means for the CB + HS group 20 min after reperfusion were also significantly higher than for the CB group for glutamine and taurine (p
< 0.05). No effects of cardioplegic technique were observed for the alanine/glutamate ratio. This is not surprising given the similar pattern of means for each amino acid separately.
3.2 Acyanotic subgroup
There were no differences between cardioplegic groups, either at the end of ischemia or 20 min after reperfusion, for any of the six amino acid markers investigated (all p values for the overall effect of cardioplegia technique >0.15). Changes over time are shown in Fig. 2
and are described quantitatively in Table 4.
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Fig. 2. Geometric means and standard errors for aspartate (A), alanine (B), branched-chain amino acids (C), glutamine (D), taurine (E) and alanine/glutamate ratio (F) in acyanotic children at the end of ischemia and 20 min after reperfusion.
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4. Discussion
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This study demonstrates that hearts of cyanotic patients respond differently to cardioplegia than hearts of acyanotic patients. In the cyanotic group, patients who were younger and had longer cross-clamp times, CB + HS preserved endogenous AAs to the greatest extent; CB on its own appeared to be better than CC but not as good as CB + HS. In the acyanotic group, patients who were older and had shorter cross-clamp times, AA levels (all p
> 0.15) and group means were similar both at the end of ischemia and after reperfusion.
4.1 Study limitations
Owing to the different characteristics of cyanotic and acyanotic patients, it is not possible to attribute these metabolic differences solely to an effect of cyanosis; age (i.e. developmental stage and its effect on ischemic tolerance and sensitivity to cardioplegia) and ischemic duration may also have played a part. For example, it may be that a difference between blood and crystalloid cardioplegia in acyanotic children would have become apparent if the ischemic time had been longer or the patients had been younger. Our previous work in adults undergoing coronary artery bypass surgery has demonstrated that CB preserves myocardial AAs compared to CC [5,6]. Nevertheless, the results of this study are consistent with our previous work in pediatric patients [4]. It is extremely difficult in a randomised trial, and perhaps academic, to distinguish individual effects of cyanosis, age and ischemic duration because these factors are inextricably linked and in part are a consequence of the pathologies affecting the patients. Although it is important from a scientific perspective to know why the hearts of cyanotic and acyanotic patients respond differently to the three cardioplegic techniques, the clinical implications of this randomised trial are nevertheless clear—CB + HS and CB are better for children with cyanosis (and the other characteristics that are associated with cyanosis) than CC; for acyanotic children, the cardioplegic technique is not critical in the context of the ischemic durations and ages of our patients.
One could also argue that because the majority of patients had relatively ‘simple’ defects (ASD, VSD, TOF) the results cannot be extended to the whole of ‘pediatric cardiac surgery’. If anything, this would make it more difficult to find a difference between the cardioplegic strategies. The fact that there were still clear differences in cardioplegic technique in the cyanotic group indicates just how vital it is to get the right technique of protection regardless of how complex the defect is. Admittedly, more complex surgery in acyanotic patients may reveal a difference between cardioplegic technique.
The power of the study was limited by the sample size which may have allowed some potentially important effects to be missed. However, most of the findings were highly statistically significant and the pattern of results consistent across the AAs investigated in the smaller cyanotic group, so it is unlikely that this was the case.
4.2 Taurine and glutamine
The non-protein ß-amino acid taurine is present at a high concentration in mammalian heart cells but at a much lower concentration in the plasma, thus creating a large concentration gradient across the sarcolemma [10]. This gradient is maintained by a Na+-taurine symport using the Na+ electrochemical gradient. A fall in tissue taurine will influence myocardial function as taurine has several important roles including membrane stabilisation, Ca2+ mobilisation, [Na]i regulation and regulation of phosphorylation of channels and transporters. Glutamine is one of the principal free intracellular amino acids in mammalian heart cells and it is important as a nitrogen donor for the biosynthesis of a number of compounds such as nucleotides and amino acids [11]. Furthermore, muscular glutamine has been shown to increase protein synthesis, decrease protein degradation and regulate glycogen metabolism [11,12].
Ischemic arrest with either crystalloid or blood cardioplegia was associated with a fall in the intracellular concentrations of taurine and glutamine. The fall in taurine is due to transport because taurine is very slowly metabolised [13,14]. The settings of the Na+-taurine transporter can be reversed to efflux taurine by the absence of the extracellular amino acid, membrane depolarisation and a rise in [Na+]i, as occurs during ischemic arrest. Unlike taurine, changes in the intracellular concentration of glutamine are likely to be the net result of protein metabolism as well as transport. Work on rat hearts has provided evidence for the absence of glutamine synthetase (thus inability of heart cells to synthesise glutamine) and the presence of glutaminase [6]. Ischemic cardiomyocytes utilise glutamate as an energy source during ischemia and our previous work has demonstrated a fall in glutamate during ischemia in pediatric hearts [4]. It is therefore plausible that the conversion of glutamine to glutamate provides the much needed substrate for the Krebs cycle during ischemia. In addition to metabolism, the transport of glutamine which is both very fast and Na+-dependent may contribute to the observed fall in this amino acid.
A further fall in taurine and glutamine occurs after 20 min reperfusion in all groups except cyanotic patients receiving a hot shot. This represents a reperfusion injury and is mirrored by changes in ATP demonstrated in our previous work [4]. It is known that soon after reperfusion, cardiomyocytes are further loaded with Na+ but most importantly they are loaded with Ca2+ in exchange for Na+, a process associated with cellular damage. The fall in taurine is another marker of a rise in [Na+]i as the changes in taurine are largely due to transport [6]. By contrast, the preservation of taurine in the group receiving a hot shot indicates that the cardiomyocytes have avoided a rise in [Na+]i. This implies that the mechanism of action of the hot shot is to allow energy stores to be used for the regeneration of ionic imbalances rather than electromechanical work during the critical first few minutes of reperfusion. By contrast, the fall in glutamine on reperfusion may have resulted from both Na+-dependent efflux and its utilisation for energy production.
4.3 Aspartate and alanine
Aspartate acts as a substrate for energy production by substituting into the Krebs cycle and, more importantly, as a participant in the malate–aspartate shuttle for balancing reducing equivalents between the cytoplasm and mitochondria [15]. The biochemical pathways for these reactions involve both the transamination of aspartate with ketoglutarate to form glutamate and oxalacetate and the transamination of pyruvate with glutamate to form alanine and ketoglutarate [16–18]. Suleiman et al. [5] demonstrated a fall in myocardial aspartate and glutamate concentrations in adult patients undergoing coronary surgery using blood and crystalloid cardioplegia. We have previously demonstrated in cyanotic pediatric patients that CB + HS significantly reduced the decrease in glutamate observed after 20 min reperfusion compared with that seen in those receiving CC and CB [4]. Similarly, there is evidence of metabolic derangement during ischemia in both acyanotic and cyanotic pediatric patients with a fall in intracellular aspartate concentrations and a rise in the alanine–glutamate ratio. On reperfusion, aspartate levels fall further with marked depletion in cyanotic patients receiving CC. This implies that crystalloid cardioplegia provides inadequate myocardial protection to cyanotic hearts leading to greater vulnerability to reperfusion injury.
Other workers have suggested that the extent of metabolic recovery after aortic cross-clamping may follow myocardial uptake of glutamate and aspartate [19]. This supports the view that exogenous glutamate and aspartate added after reperfusion may enhance myocardial metabolism. Our cardioplegic strategy did not include amino acid enrichment and it is plausible to suggest that we would have seen better metabolic recovery had this been used [20].
4.4 Branched chain amino acids
There were no clinically significant changes in the concentrations of the branched chain AAs with time in either group implying that changes in other AAs are not related to protein breakdown or synthesis [21].
Despite the metabolic advantages of warm cardioplegic reperfusion in cyanotic patients, our previous work has demonstrated no differences in clinical outcomes, indicating that the changes seen during ischemia were probably completely reversible [4]. However, clinical outcomes are a crude measure of the efficacy of myocardial protection; functional outcomes, which we did not measure, are a better indicator of this. Because metabolite preservation has been shown to correlate with better functional recovery, it might be that CB and terminal warm blood cardioplegic reperfusion allow for improved post-operative cardiac function [22].
In summary, for cyanotic patients, who tend to be younger and have longer cross-clamp times, cold blood cardioplegia supplemented with a hot shot preserves myocardial free AAs better than cold crystalloid cardioplegia; cold blood cardioplegia on its own gives an intermediate effect. In acyanotic patients, AA levels (all p
> 0.15) and group means were similar both at the end of ischemia and after reperfusion.
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Acknowledgments
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British Heart Foundation, National Heart Research Fund and Garfield Weston Trust supported this work. We would like to thank Mark Ginty and Svitlana Korolchuk for performing the biochemical analyses and the pediatric nursing staff for their support.
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References
|
|---|
- Riva E, Hearse DJ. The developing myocardium. New York: Futura Publishing Company, Inc.; 1991.
- Baker JE, Boerboom LE, Olinger GN. Is protection of ischemic neonatal myocardium by cardioplegia species dependent?. J Thorac Cardiovasc Surg 1990;99:280-287.[Abstract]
- Ostadal B, Ostadalova I, Dhalla NS. Development of cardiac sensitivity to oxygen deficiency: comparative and ontogenetic aspects. Phys. Rev. 1999;79(3):635-659.[Abstract/Free Full Text]
- Modi P, Suleiman M-S, Reeves B, Pawade A, Parry A, Angelini GD, Caputo M. Myocardial metabolic changes during paediatric cardiac surgery: a randomised study of three cardioplegic techniques. J Thorac Cardiovasc Surg 2004;128(1):67-75.[Abstract/Free Full Text]
- Suleiman M-S, Dihmis WC, Caputo M, Angelini GD, Bryan AJ. Changes in the myocardial concentration of glutamate and aspartate during coronary artery surgery. Am J Physiol 1997;272(3 (Pt 2)):H1063-H1069.
- Suleiman M-S, Moffat A, Dihmis WC, Caputo M, Hutter JA, Angelini GD, Bryan AJ. Effect of ischaemia and reperfusion on the intracellular concentration of taurine and glutamine in the hearts of patients undergoing coronary artery surgery. Biochem Biophys Acta 1997;1324:223-231.[Medline]
- Julia P, Young H, Buckberg GD, Kofsky E, Bugyi HI. Studies of myocardial protection in the immature heart. II. Evidence for importance of amino acid metabolism in tolerance to ischemia. J Thorac Cardiovasc Surg 1990;100:888-895.[Abstract]
- Imura H, Caputo M, Parry A, Pawade A, Angelini GD, Suleiman M-S. Age-dependent and hypoxia-related differences in myocardial protection during pediatric open-heart surgery. Circulation 2001;103(11):1551-1556.[Abstract/Free Full Text]
- Caputo M, Dihmis W, Bryan AJ, Suleiman M-S, Angelini GD. Warm blood hyperkalaemic reperfusion (hot shot) prevents myocardial substrate derangement in patients undergoing coronary artery bypass surgery. Eur J Cardiothoracic Surg 1998;13:559-564.
- Chapman RA, Suleiman M-S, Earm YE. Taurine and the heart. Cardiovasc Res 1993;27:358-363.[Free Full Text]
- Rennie MJ, Trados L, Khogali S, Ahmed A, Taylor P. Glutamine transport and its metabolic effects. J Nutr 1994;124(Suppl):S1503-S1508.[Abstract/Free Full Text]
- MacLennan PA, Smith K, Weryk B, Watt PW, Rennie MJ. Inhibition of protein breakdown by glutamine in perfused rat skeletal muscle. FEBS Lett 1988;237:133-136.[CrossRef][Medline]
- Suleiman M-S. New concepts in the cardioprotective action of magnesium and taurine during the calcium paradox and ischemia of the heart. Magnesium Res 1994;7:295-312.
- Huxtable RJ. Physiological actions of taurine. Physiol Rev 1992;72:101-163.[Free Full Text]
- Safer B. The metabolic significance of the malate–aspartate cycle in heart. Circ Res 1975;37(5):527-533.[Free Full Text]
- Taegtmeyer H. Metabolic responses to cardiac hypoxia: increased production of succinate by rabbit papillary muscle. Circ Res 1978;43:808-815.[Free Full Text]
- Sanborn T, Gavin W, Berkowitz S, Perille T, Lesch M. Augmented conversion of aspartate and glutamate to succinate during anoxia in rabbit heart. Am J Physiol 1979;237:H535-H541.
- Freminet A, Leclerc L, Poyart C, Huel C, Gentil M. Alanine and succinate accumulation in the perfused rat heart during hypoxia. J Physiol (Paris) 1980;76(2):113-117.[Medline]
- Svedjeholm R, Hakanson E, Vanhanen I. Rationale for metabolic support with amino acids and glutamate and glucose-insulin-potassium (GIK) in cardiac surgery. Ann Thorac Surg 1995;59:S15-S22.[CrossRef][Medline]
- Allen BS. Pediatric myocardial protection: where do we stand?. J Thorac Cardiovasc Surg 2004;128(1):11-13.[Free Full Text]
- Mackenzie B, Ahmed A, Rennie MJ. Muscle amino acid metabolism and transport. In: Kilberg MS, Haussinger D, editors. Mammalian amino acid transport. New York: Plenum Press; 1992. pp. 195-231.
- Hammon JW, Graham TP, Boucek RJ, Parrish MD, Merrill WH, Bender HW. Myocardial adenosine-triphosphate content as a measure of metabolic and functional myocardial protection in children undergoing cardiac operation. Ann Thorac Surg 1987;44(5):467-470.[Abstract]
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Abstract
1. Introduction
