Basal Metabolic State of Hearts of Patients With Congenital Heart Disease: The Effects of Cyanosis, Age, and Pathology

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1 Basal Metabolic State of Hearts of Patients With Congenital Heart Disease: The Effects of Cyanosis, Age, and Pathology Paul Modi, FRCS, M.-Saadeh Suleiman, DSc, Barnaby C. Reeves, PhD, Ash Pawade, FRCS, Andrew J. Parry, FRCS, Gianni D. Angelini, FRCS, and Massimo Caputo, MD Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom Background. Experimental models have established numerous myocardial metabolic changes with chronic hypoxia and maturation. We conducted this study to specifically look at the effects of cyanosis, age, and pathology upon the basal metabolic state of the immature human heart. Methods. One hundred and eighty-one pediatric patients (37 cyanotic, 144 acyanotic) undergoing open heart surgery were recruited. A myocardial biopsy was collected before ischemia and analyzed for adenine nucleotides, purines, and lactate. The effect of cyanosis was estimated by an analysis of age-matched pairs of children with either ventricular septal defects or tetralogy of Fallot, and by multiple regression modeling. The effects of age and pathology were estimated in acyanotic children also by multiple regression modeling (adjustments were made for baseline differences). Results. The only effect of cyanosis was for lactate where the paired t test, and unadjusted and adjusted regression analyses were all consistent (ranging from 1.33 to 1.48 times higher in cyanotic than acyanotic children). The concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) declined with age, whereas the ATP/ ADP ratio increased; these associations remained significant even in the adjusted regression analysis. None of the effects of acyanotic pathology were highly significant (p < 0.01), implying that few important metabolic differences were attributable to pathology. Conclusions. Cyanosis and age are important factors that determine the basal metabolic state of the pediatric heart. Cyanotic patients have higher myocardial lactate concentrations, whereas young age is associated with lower ATP/ADP ratios and higher adenine nucleotide levels. (Ann Thorac Surg 2004;78:1710 6) 2004 by The Society of Thoracic Surgeons We have recently demonstrated that cyanotic infants who undergo open heart surgery sustain greater myocardial reperfusion injury and have a worse clinical outcome than acyanotic children [1]. The injury could occur as a result of intraoperative factors, but may also be due to preoperative factors, such as the basal myocardial metabolic state, where a stressed heart may be more vulnerable to subsequent ischemic injury [2]. However, little is known about the basal myocardial metabolic state or how it is affected by cyanosis, age, and pathology. Indeed, the current cardioprotective techniques that are used in pediatric cardiac surgery, which in fact were designed to protect adult hearts, do not take into account differences in basal metabolism. Inherent in a rational approach to myocardial protection must be an understanding of the baseline metabolic profile even before the stresses of cardioplegic arrest are scrutinized. Accepted for publication May 3, Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26 28, Address reprint requests to Dr Modi, Derriford Hospital, Derriford, Plymouth PL6 9DH, UK; paulmodi@doctors.org.uk. Our recent work has shown that pathology affects the metabolic state in adult hearts. Hypertrophic hearts from patients with aortic stenosis have higher concentrations of adenosine triphosphate (ATP) but lower concentrations of lactate, branched-chain amino acids, and alanine than the hearts of patients with ischemic heart disease [3]. These findings imply important differences in energy metabolism and protein turnover between the two pathologies. Experimental models of the immature heart have also established numerous metabolic changes with chronic hypoxia and maturation that affect its inherent tolerance to ischemia [2, 4 6]. A limited number of clinical studies have used different techniques of myocardial protection in an attempt to assess metabolism (eg, adenine nucleotides) during ischemia and reperfusion in the immature pediatric heart, but none have specifically focused on the markers of metabolic stress that exist before ischemia [7]. The identification of metabolically stressed subsets of patients would therefore be useful in predicting outcome and may also influence intraoperative cardioprotective practice. The aim of this study was therefore to determine the 2004 by The Society of Thoracic Surgeons /04/$30.00 Published by Elsevier Inc doi: /j.athoracsur

2 Ann Thorac Surg MODI ET AL 2004;78: METABOLITES IN CONGENITAL HEART DISEASE Abbreviations and Acronyms ADP adenosine diphosphate AMP adenosine monophosphate ASD atrial septal defect ATP adenosine triphosphate CPB cardiopulmonary bypass Hb hemoglobin PA pulmonary atresia RV right ventricular Sao 2 arterial oxygen saturation TOF tetralogy of Fallot VSD ventricular septal defect preischemic basal metabolic state of the immature pediatric heart with congenital heart disease and investigate the effects of cyanosis, age and pathology upon this by measuring a variety of important markers of metabolic stress in myocardial biopsies. Material and Methods Children undergoing elective repair of congenital heart defects between June 2000 and December 2001 at the Royal Hospital for Children, Bristol, were recruited. The study was approved by the local Hospital Ethics Committee, and written informed consent was obtained from the parents/guardians of all patients. Anesthetic technique was standardized, as reported previously [1]. Cardiopulmonary bypass (CPB) was established between ascending aortic and bicaval cannulas with moderate systemic hypothermia (28 to 32 C). Immediately before cross-clamping the aorta, a myocardial biopsy (mean weight SD, mg) was collected from the anterior wall of the right ventricle with an 18G 6-cm Trucut biopsy needle (Allegiance Healthcare, McGraw, IL). The specimen was immediately frozen in liquid nitrogen until processing for the analysis of metabolites. Adenine nucleotides and purines in the neutralized extract were separated and quantified by use of high-performance liquid chromatography based on previous reports [8]. Lactate was determined by use of a diagnostic kit from Sigma (Poole, Dorset, UK). Protein determination was done according to the Lowry method by using a protein determination kit from Sigma with bovine serum albumin as a standard. Metabolite concentrations were calculated as nmol per mg of protein content. Statistical Analysis Data from the preischemic biopsy of 103 patients in a previous study of myocardial protection were included, as the protocol and timing of the biopsy collection were identical [9]. Medians and interquartile ranges or frequencies were used to summarize descriptive demographic and clinical data. Comparative analyses were computed to estimate the effects of cyanosis, age, and diagnosis on metabolic markers, as far as possible taking into account potential confounding factors. Data for metabolic markers were transformed by taking natural logarithms to normalize their distributions (raw data were positively skewed). Effects are therefore expressed as relative changes in geometric means. Two sets of analyses were done to estimate the effect of cyanosis (defined as a resting arterial oxygen saturation 90%). First, pairs of age-matched children with ventricular septal defects (VSDs, acyanotic) and children with either tetralogy of Fallot (TOF) or pulmonary atresia (PA) with VSD (PA plus VSD) (cyanotic) were created without reference to biochemical data; age matching was within 1 month for children aged less than 3 years, and within 3 months for older children. The effects of cyanosis were estimated by paired t tests. Second, multiple regression modeling was used to analyze the data for all children aged between 1 month and 4 years, this being the age range over which both cyanotic and acyanotic children were reasonably well represented. The effects of age and pathology were estimated only in acyanotic children, also by multiple regression modeling. Multiple regression modeling was completed interactively. Unadjusted models for cyanosis and age included just these variables. Adjusted models also considered resting arterial oxygen saturation (Sao 2 ), hemoglobin (Hb), and diagnosis; these variables were included if they significantly improved the fit of the model or in the case of diagnosis, if they were of primary interest to the analysis. Weight was not considered, because it was very strongly correlated with age. The exploratory nature of the analysis and the number of metabolic markers of interest resulted in a large number of statistical comparisons. No correction was made for multiple comparisons, but our interpretation of the findings takes into account the consistency of the findings and their magnitude as well as their statistical significance. All data were analyzed using Stata version 7 (Stata Corporation, College Station, TX). Results 1711 Of the 181 children who were recruited, 113 were aged 4 years or younger, and only one was less than 1 month old. Thirty-seven patients (20%) were cyanotic and were significantly younger, with lower Sao 2 and higher Hb concentrations (Table 1); all had TOF or PA plus VSD. The protein content of myocardial tissue was similar in cyanotic and acyanotic hearts (mean SEM, 9.9% 1.3% vs 9.6% 0.2% of wet weight, respectively, p 0.68). The metabolic data are summarized in Table 2 in natural units. Effects of Cyanosis Because of differences in age, only 22 pairs of cyanotic (TOF or PA plus VSD) and acyanotic children (VSD) could be age-matched. Paired t tests found no significant effects of cyanosis, but the estimate for lactate suggested this metabolic marker was higher in cyanotic than acyanotic children (33% higher, ie, 1.33 times, 95% CI 0.66 to 2.59, p 0.40). Adjusted and unadjusted estimates of the effects of CARDIOVASCULAR

3 1712 MODI ET AL Ann Thorac Surg METABOLITES IN CONGENITAL HEART DISEASE 2004;78: Table 1. Patient Characteristics Acyanotic Cyanotic n Age (months) 42.0 (7.0 to 98.5) 13.0 (6.8 to 21.0) Weight (kg) 14.1 (6.7 to 23.8) 8.7 (7.0 to 10.9) Sao 2 (%) 98.0 (97.0 to 99.0) 79.5 (72.0 to 85.0) Hb (g/dl) 12.3 (11.5 to 13.3) 14.9 (13.8 to 16.3) Pathology ASD 43 (33.3) VSD 48 (29.9) TOF 11 (7.6) 28 (75.7) PA VSD 9 (24.3) Other 42 (29.2) Other acyanotic diagnoses included: aortic stenosis/regurgitation (n 24); complete atrioventricular septal defect (n 6); mitral/tricuspid regurgitation (n 6); pulmonary vein anomalous drainage/stenosis (n 3); miscellaneous (n 3). Data are medians and interquartile ranges, or number (%) of observations. ASD atrial septal defect; PA pulmonary atresia; TOF tetralogy of Fallot; VSD ventricular septal defect. cyanosis are shown in Table 3 for children aged between 1 month and 4 years. Unadjusted estimates are based on the data for all children in this age range (n 112). Adjusted estimates were restricted to diagnoses of VSD (acyanotic) and TOF/PA plus VSD (cyanotic) in order to control as much as possible for the effect of diagnosis (n 74). The effect estimates for lactate from the paired t test, and the unadjusted and adjusted regression analyses are all remarkably consistent (ranging from 1.33 times higher to 1.48 times higher in cyanotic than acyanotic children). However, the effect was only significant for the unadjusted analysis because of the larger sample size when children with all diagnoses were included. The only other metabolite that tended to be associated with cyanosis was adenosine, with effect estimates ranging from 1.24 times to 1.34 times higher (p 0.11). Effects of Age and Pathology The effects of age and pathology in children with acyanotic diagnoses are shown in Tables 4 and 5, respectively. The concentrations of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) declined with age, whereas the ATP/ADP ratio increased with age; these associations remained significant after adjustment for Sao 2, Hb, and diagnosis (as categorized in Table 1). Lactate and inosine also tended to decline with age, but in the adjusted estimates these changes were not significant. The effects of different acyanotic pathologies on the basal metabolic state were expressed in relation to children with VSDs (the largest diagnostic group). Excluding the effects of hypoxanthine, which were very variable across children (as evidenced by the wide confidence intervals), effect sizes ranged from a reduction in AMP of 0.63 times (p 0.04) to an increase in the ATP/ADP ratio of 1.40 (p 0.02) in children with acyanotic TOF. However, most effects were much smaller (two thirds of 27 comparisons were within 20% of the levels for children with VSDs), and none were highly significant (p 0.01), implying that within the acyanotic group, few important metabolic differences were attributable to pathology. Comment This study demonstrates that cyanosis was associated with higher myocardial lactate concentrations but with no differences in adenine nucleotide or purine content. The concentrations of ATP, ADP, and AMP declined with age, whereas the ATP/ADP ratio increased; these associations remained significant even in the adjusted regression analysis. None of the effects of acyanotic pathology were highly significant (p 0.01), implying that there were few important metabolic differences between the acyanotic pathologies represented in the study population. Cyanosis Few authors have looked at the influence of cyanosis upon the basal metabolic state of the immature human heart. We assessed this using two sets of analyses (agematched pairs with comparable diagnoses (VSD vs TOF/PA plus VSD) and multiple regression modeling) and found similar results with each. As our previous work has shown [1], lactate levels in cyanotic hearts were higher, indicating a greater dependence on anaerobic metabolism for ATP production in keeping with the reduction in oxygen availability. This suggests that these hearts are more metabolically stressed and may develop tissue acidosis that leads to irreversible myocardial injury sooner during ischemia than do acyanotic hearts. This agrees with work on chronically hypoxic animal models [2, 4] and cyanotic human hearts [1, 10] that shows an accelerated depletion of ATP during ischemia and greater injury upon reperfusion. Others have suggested that chronic exposure to hypoxia increases the tolerance of the immature rabbit heart to ischemia [5]; however, as species differences are known to exist, the immature human heart may differ [11]. Table 2. Means for Metabolic Data in Natural Units Acyanotic Cyanotic n Lactate ATP ADP AMP Adenosine Inosine Hypoxanthine ATP/ADP ratio Data are mean SEM. Units of metabolic data are nmol per mg protein. No statistical comparisons by cyanosis were made. ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate.

4 Ann Thorac Surg MODI ET AL 2004;78: METABOLITES IN CONGENITAL HEART DISEASE Table 3. Unadjusted and Adjusted Estimates of the Effect of Cyanosis on Metabolic Markers in Children Aged 1 Month to 4 Years Unadjusted Estimates a Effect of Cyanosis a 95% CI p Adjusted Estimates 1713 Effect of Cyanosis b 95% CI p CARDIOVASCULAR Lactate to to ATP to to ADP to to AMP to to Adenosine to to Inosine to to Hypoxanthine to to ATP/ADP ratio to to a Unadjusted estimates represent the effect of cyanosis, only taking account of age, for all children aged 1 month to 4 years (n 112). Effect of cyanosis represents the relative increase or decrease for cyanotic children compared with acyanotic children, i.e., the unadjusted estimate for lactate (1.48) means that the geometric mean for cyanotic children was 1.48 times (48% greater) than for acyanotic children. b Adjusted estimates represent the effect of cyanosis including other factors (Hb, Sao 2 ) that were significant predictors of the metabolic markers, and restricting the analysis to children aged 1 month to 4 years with diagnoses of VSD (acyanotic) and tetralogy of Fallot or pulmonary atresia and VSD (cyanotic) to take account of diagnosis (n 74). Effect of cyanosis should be interpreted in the same way as for unadjusted estimates. ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; VSD ventricular septal defect. It is beyond the scope of this work to speculate on the appropriate technique of myocardial protection for cyanotic hearts, but it is plausible to suggest that techniques that have been shown to resuscitate metabolically stressed hearts (eg, blood cardioplegia, amino acid cardioplegic enrichment) [12] may be more effective in cyanotic hearts, particularly as the immature heart has a greater dependence on amino acid transamination for energy generation during ischemia [13]. This agrees with our recent work that demonstrates superior myocardial protection with blood cardioplegia and terminal warm blood cardioplegic reperfusion in cyanotic hearts [9]. There was no difference in adenine nucleotide content between acyanotic and cyanotic hearts indicating that anaerobic metabolism is able to fully meet the energy requirements of the cyanotic myocardium at rest. Silverman and colleagues created a chronically hypoxic canine model by anastomosing the left atrium proximal to a banded pulmonary artery and also noted that compared with nonhypoxic controls, there was no difference in base line ATP or creatine phosphate levels [2]. Imura and colleagues looked at the basal metabolic state in 58 patients who were undergoing open-heart surgery and also noted that the ATP level was not significantly lower in cyanotic patients [1]. On the other hand, Najm and colleagues studied the effect of the degree of cyanosis on ATP levels in 48 patients who were undergoing repair of tetralogy and noted a stepwise Table 4. Unadjusted and Adjusted Estimates of the Effect of Increasing Age on Metabolic Markers in Acyanotic Children Unadjusted Estimates Adjusted Estimates Change per yr a 95% CI p Change per yr b 95% CI p Lactate to to ATP: age to to age c to to ADP to to AMP to to Adenosine to to Inosine to to Hypoxanthine to to ATP/ADP ratio to to a Unadjusted estimates represent the effect of age without taking account of other factors, for all acyanotic children (n 144). Change per year is expressed in the same way as in Table 1, but in this case represents the relative increase or decrease per year of age, i.e. the unadjusted estimate for lactate (0.97) means that the geometric mean decreases by 0.97 times (1 0.97, or 3% decrease) for every additional year of age. b Adjusted estimates are derived from multivariable regression models, including other factors (Hb, Sao 2 and diagnosis) that were significant predictors of the metabolic markers. The adjusted estimate for ADP (0.98) means that the geometric mean decreases by 0.98 times (1 0.98, or 2% decrease) for every additional year of age. c For ATP only, the effect of age was best modelled by including a quadratic (age 2 ) term in the regression, i.e. the relationship with age was curvilinear, in this case the decrease with age accelerated with increasing age. ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; Hb hemoglobin.

5 1714 MODI ET AL Ann Thorac Surg METABOLITES IN CONGENITAL HEART DISEASE 2004;78: Table 5. Adjusted Estimates of the Effects of Diagnosis, Compared with VSD, on Metabolic Markers in Acyanotic Children ASD vs VSD a 95% CI p acytof vs VSD b 95% CI p Other vs VSD c 95% CI p d p e Lactate to to to ATP to to to ADP to to to AMP to to to Adenosine to to to Inosine to to to Hypoxanthine to to to ATP/ADP ratio to to to a Adjusted estimates represent the relative increase or decrease in the geometric mean of the metabolite for ASD compared with VSD. b Adjusted estimates represent the relative increase or decrease in the geometric mean of the metabolite for acytof compared with VSD. c Adjusted estimates represent the relative increase or decrease in the geometric mean of the metabolite for other acyanotic diagnoses compared with VSD. d Significance of the effect of the specific diagnosis in the regression model. e Significance of the overall effect of including diagnosis, i.e. all four diagnostic categories combined, in the regression model. acytof acyanotic tetralogy of Fallot; ADP adenosine diphosphate; AMP adenosine monophosphate; ATP adenosine triphosphate; ASD atrial septal defect; Other acyanotic diagnoses including aortic stenosis/regurgitation (n 24); complete atrioventricular septal defect (n 6); mitral/tricuspid regurgitation (n 6); pulmonary vein anomalous drainage/stenosis (n 3); miscellaneous (n 3). decrease in ATP concentrations with increasing desaturation (for Sao 2 of 90% to 100%, 80% to 89%, and 65% to 79%, ATP concentrations were , , and mol/g dry weight, respectively) [14]. Age Younger age was associated with higher levels of adenine nucleotides and inosine but lower ATP/ADP ratios. Jarmakani and colleagues demonstrated that myocardial high-energy phosphate content in the neonatal rabbit was as high, if not higher, than in the adult [15], and in comparison to the levels we have previously reported in ischemic and hypertrophic adult hearts, this seems to be the case [3]. This greater ATP content in neonates may occur as a consequence of lower energy demands with decreased rates of utilization [16], greater glycolytic ATP production [17], decreased efflux of ATP or its degradation products [18], or increased rates of resynthesis [19]. On the other hand, Lofland and colleagues noted that there were no differences in baseline ATP levels between patients younger or older than 18 months [20]. However, they related higher levels of AMP and inosine in patients younger than 18 months old to a deficiency of 5 nucleotidase, a regulatory enzyme that dephosphorylates AMP to adenosine and may well persist beyond the neonatal period. Morphologic studies have shown a deficiency in immature myocardial cells of T tubules where 5 -nucleotidase is normally concentrated in the adult heart [21]. Our data demonstrate similar findings, with a decrease in AMP and inosine with increasing age. Matherne and colleagues compared the basal bioenergetic state of hearts from 2- to 4-week-old rabbits to that from 3- to 4-month-old rabbits [22]. Basal ATP concentrations were comparable in the two age groups; however, the resting cytosolic phosphorylation ratio (ie, ATP/ ADP ratio) was lower in immature than in mature hearts, as we have demonstrated. As the ability of mitochondria to phosphorylate ADP to ATP is one of the most important determinants of resistance to ischemia, this may offer an explanation to reports suggesting that the immature human heart is less resistant to ischemia than its more mature counterpart [1, 22, 23]. Pathology Many infants with congenital heart disease are exposed to volume overload secondary to a large left-to-right shunt, such as is seen with a ventricular septal defect. Riva and colleagues suggested that volume overloading may result in unfavorable metabolic changes in the myocardium [6]. However, few important differences were attributable to pathology, and the greater volume load that was associated with VSDs did not seem to alter the basal metabolic state compared with hearts with atrial septal defects (ASDs). Additionally, acyanotic hearts with TOF had lactate concentrations similar to hearts with VSDs rather than to cyanotic hearts with TOF. This implies that cyanosis and age are the most important factors that determine the basal metabolic state of the pediatric heart. Implications for Cardiac Surgery The final tally of injury after cardiac surgery is due to a combination of hypoxia-reoxygenation upon commencement of CPB [24] and ischemia-reperfusion caused by aortic clamping modified by the inherent tolerance of the myocardium to these factors [2]. The information presented in this paper identifies hearts that are metabolically stressed and may help in formulating cardioprotective strategies. For example, techniques such as substrate enrichment (eg, aspartate and glutamate) may be most beneficial in cyanotic and infant hearts. Study Limitations Several confounding factors could have affected the metabolic markers. First, there are two periods during which the cyanotic hearts may have undergone a reoxygenation injury: at the induction of anesthesia when the patient is ventilated with high concentrations of oxygen and at the

6 Ann Thorac Surg MODI ET AL 2004;78: METABOLITES IN CONGENITAL HEART DISEASE commencement of CPB when the priming fluid is hyperoxic. Although acyanotic hearts undergo the same injury, this has been demonstrated to occur earlier and be greater in cyanotic hearts and may have affected metabolite concentrations [24]. Nevertheless, as stated in our aims, we sought to measure metabolite levels immediately before ischemic arrest. Second, the metabolic state of hearts with TOF and VSDs may be affected by the degree of right ventricular (RV) hypertrophy. In children with TOF, cyanosis and hypertrophy go hand-in-hand, whereas in children with VSDs, the degree of RV hypertrophy is much more variable and depends on the RV pressure. However, the consistency of the size of the effect of cyanosis on lactate concentrations from both the multiple regression modeling and the paired t test suggests that this is unlikely to affect the interpretation of the results. Besides, data from adults have suggested that hypertrophic hearts have lower lactate levels than ischemic hearts, an effect opposite in direction to that seen in our immature hearts with TOF [3]. Third, our study had limited power to detect a metabolic difference that was due to cyanosis. Given the unequal sample sizes of cyanotic and acyanotic children (about 1:2 for the analyses with the largest sample size), the unadjusted regression analyses had 80% power to detect a standardized difference of about 0.6 between groups at a significance level of 0.05 (2-tailed), ie, a moderate-to-large effect. The power of the paired t tests was very much lower. In summary, this study demonstrates that cyanosis and age are important factors determining the basal metabolic state of the pediatric heart. Cyanosis is associated with higher myocardial lactate concentrations, whereas young age is associated with lower ATP/ADP ratios and higher adenine nucleotide levels. The British Heart Foundation and The National Heart Research Fund supported this work. We would like to thank Anne Moffat, Svitlana Korolchuk, and Mark Ginty for expert technical assistance. References 1. 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): Silverman NA, Kohler J, Levitsky S, Pavel DG, Fang RB, Feinberg H. Chronic hypoxemia depresses global ventricular function and predisposes to the depletion of high-energy phosphates during cardioplegic arrest implications for surgical repair of cyanotic congenital heart defects. Ann Thorac Surg 1984;37(4): Suleiman M-S, Caputo M, Ascione R, et al. Metabolic differences between hearts of patients with aortic valve disease and hearts of patients with ischaemic disease. J Mol Cell Cardiol 1998;30: Fujiwara T, Kurtts T, Anderson W, Heinle J, Mayer JE. Myocardial protection in cyanotic neonatal lambs. J Thorac Cardiovasc Surg 1988;96: Baker EJ, Boerboom LE, Olinger GN, Baker JE. Tolerance of 1715 the developing heart to ischemia: impact of hypoxemia from birth. Am J Physiol 1995;268:H Riva E, Hearse DJ. The developing myocardium. New York: Futura Publishing Company, Inc, Caputo M, Modi P, Imura H, et al. Cold blood versus cold crystalloid cardioplegia for repair of ventricular septal defects in pediatric heart surgery: a randomized controlled trial. Ann Thorac Surg 2002;74(2): Smolensky RT, Lachno DR, Ledingham SJM, Yacoub MH. Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J Chromatogr 1996;527: Modi P, Suleiman M-S, Reeves B, et al. Myocardial metabolic changes during pediatric cardiac surgery: a randomized study of three cardioplegic techniques. J Thorac Cardiovasc Surg 2004 [in press]. 10. Del Nido RJ, Mickle DAG, Wilson GJ, et al. Inadequate myocardial protection with cold cardioplegic arrest during repair of tetralogy of Fallot. J Thorac Cardiovasc Surg 1988; 95(2): Baker JE, Boerboom LE, Olinger GN. Is protection of ischemic neonatal myocardium by cardioplegia species dependent? J Thorac Cardiovasc Surg 1990;99: Bolling K, Kronon M, Allen BS, Wang T, Ramon S, Feinberg H. Myocardial protection in normal and hypoxically stressed neonatal hearts: the superiority of blood versus crystalloid cardioplegia. J Thorac Cardiovasc Surg 1997;113(6): 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: Najm HK, Wallen WJ, Belanger MP, et al. Does the degree of cyanosis affect myocardial adenosine triphosphate levels and function in children undergoing surgical procedures for congenital heart disease? J Thorac Cardiovasc Surg 2000; 119(3): Jarmakani JM, Nagatomo T, Langer GA. The effect of calcium and high-energy phosphate compounds on myocardial contracture in the newborn and adult rabbit. J Mol Cell Cardiol 1978;10: Young HH, Shimizu T, Nishioka K, Nakanishi T, Jarmakani JM. Effect of hypoxia and reoxygenation on mitochondrial function in neonatal myocardium. Am J Physiol 1983;245: H Hoerter JA, Opie LH. Perinatal changes in glycolytic function in response to hypoxia in the incubated or perfused rat heart. Biol Neonate 1978;33: Achterberg PW, Nieukoop AS, Schousten B, DeJong JW. Different ATP-catabolism in reperfused adult and newborn rat hearts. Am J Physiol 1988;254:H Murphy CE, Salter DR, Morris JJ, Goldstein JP, Wechsler AS, Abd-Elfattah AS. Age-related differences in adenine nucleotide metabolism during in vivo global ischemia. Surg Forum 1986;37: Lofland GK, Abd-Elfattah AS, Wyse R, de Leval M, Stark J, Wechsler AS. Myocardial adenosine nucleotide metabolism in pediatric patients during hypothermic cardioplegic arrest and normothermic ischemia. Ann Thorac Surg 1989;47: Carlsson E, Kjorell U, Thornell LE, Lambertsson A, Strehler E. Differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart. Eur J Cell Biol 1982;27(1): Matherne GP, Ely SW, Headrick JP. Maturational differences in bioenergetic state and purine formation during supply and demand ischemia. J Mol Cell Cardiol 1996;28: Taggart DP, Hadjinikolas L, Wong K, Yap J, et al. Vulnerability of paediatric myocardium to cardiac surgery. Heart 1996;76: Modi P, Imura H, Caputo M, et al. Cardiopulmonary bypassinduced myocardial reoxygenation injury in cyanotic pediatric patients. J Thorac Cardiovasc Surg 2002;124(5): CARDIOVASCULAR

7 1716 MODI ET AL Ann Thorac Surg METABOLITES IN CONGENITAL HEART DISEASE 2004;78: DISCUSSION DR DOUGLAS D. PAYNE (Boston, MA): Is it possible that the elevated lactates in the myocardial biopsy simply reflected systemic levels of lactate? And were levels of lactate measured in the blood? DR MODI: No, we did not measure lactate in the blood, just in the myocardial biopsies. DR CHARLES FRASER, JR (Houston, TX): Perhaps I missed it in your description of your methodology, but I was a little bit interested in how you handle the biopsies. Back some years ago, I had an opportunity to measure high-energy phosphate metabolism in transplanted hearts in dogs, and the rapidity of degradation of the high-energy phosphates is measured in milliseconds. And so if there is a difference in handling amongst individuals, it may cause some variability in your data, which eliminates the possibility of finding a true difference. So I was wondering about that. Did you freeze clamp these biopsies or how were they handled? DR MODI: An excellent point, thank you for bringing that up. After the biopsy was taken, it was immediately transferred into a small plastic vial and snap frozen in liquid nitrogen. DR SCOTT M. BRADLEY (Charleston, SC): Do the tetralogy patients have higher myocardial lactate because they are cyanotic, or because they have RV pressure load and hypertrophy? I ask that question particularly because it seemed that when you looked at your entire patient population, there was a significant difference in lactate levels. But when you limited it to tetralogy- VSD matched pairs, there was less of a difference. Obviously, some of the VSD patients may have had an RV pressure load as well. Were you able to tease that out of your data? DR MODI: From the data we have, it is impossible to say exactly what it is due to, whether it is due to anaerobic metabolism, which one would expect, or whether it is due to stress on the right ventricle. I suspect, however, it is a combination of a few things. DR BRADLEY: So you did not divide the patients up in terms of what their RV pressures were going into surgery? DR MODI: No, sir.