Ann Thorac Surg 2006;81:943-949
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Free Amino Acids in Hearts of Pediatric Patients With Congenital Heart Disease: The Effects of Cyanosis, Age, and Pathology
Paul Modi, FRCS,
M.-Saadeh Suleiman, DSc,
Barnaby C. Reeves, DPhil,
Ash Pawade, FRCS,
Andrew J. Parry, FRCS,
Gianni D. Angelini, FRCS,
Massimo Caputo, MD
*
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom
Accepted for publication August 25, 2005.
* Address correspondence to Dr Caputo, Bristol Heart Institute, Level 7, Bristol Royal Infirmary, Bristol, UK BS2 8HW United Kingdom (Email: m.caputo{at}bristol.ac.uk).
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Abstract
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BACKGROUND: The immature heart has a much greater dependence than the adult heart on amino acid transamination in determining its ischemic tolerance. Compared with adult hearts, experimental models of the immature heart have quantified higher resting concentrations of free amino acids (AA) which are depleted by acute hypoxia. However, we have found no clinical studies that have looked at the free AA profile of the immature human heart or the effects of cyanosis, age, and pathology upon this.
METHODS: One hundred eighty-one pediatric patients (37 cyanotic, 144 acyanotic) undergoing open-heart surgery were recruited. Myocardial biopsies were collected prior to ischemia and analyzed for free AAs (eg, glutamate, aspartate) using high-performance liquid chromatography. The effects of cyanosis, age, and pathology on amino acid concentrations were estimated by multiple regression modeling with and without controlling for diagnosis; the effects of age and pathology were estimated only in acyanotic children.
RESULTS: Alanine concentrations were about 20% higher in cyanotic than acyanotic patients (p = 0.04). Cyanosis was not associated with any other amino acid levels. In acyanotic patients, after controlling for diagnosis, concentrations of glutamate, aspartate, and alanine decreased from birth to about 8 to 10 years, then started to increase again (p < 0.05 for both linear and quadratic terms); concentrations of taurine and the branched chain AAs decreased steadily with increasing age (p < 0.05). There were significant effects of pathology on glutamate (p = 0.006), glutamine (p = 0.003), and branched chain AA (p = 0.004) levels.
CONCLUSIONS: There is no evidence that chronic hypoxia depletes endogenous AAs. Young age is associated with higher resting AA levels.
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Introduction
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Amino acids (AA) are largely thought of as building blocks for protein synthesis rather than important intermediate metabolites. However, many AAs such as glutamate and aspartate play an important metabolic role in the ischemic myocardium, where they can be used as energy substrate under anaerobic conditions. For example, the inclusion of these AAs in the perfusate improves the metabolic state of ischemic and hypoxic animal hearts [1] and their addition to blood cardioplegic solutions, both at induction and reperfusion, maintains high-energy phosphate levels by replenishing Krebs cycle intermediates and improves postischemic functional recovery [2]. Experimental models have suggested that immature hearts have higher resting levels of glutamate and aspartate compared with mature hearts and an increased resistance to ischemia that is related to amino acid utilization by transamination and substrate level phosphorylation [3, 4]. A limited number of clinical studies have attempted to assess metabolism, in particular adenine nucleotide levels, during ischemia and reperfusion in the immature pediatric heart using different techniques of cardioplegic myocardial protection [5, 6] but we have found none that have specifically focused on amino acids or their levels prior to ischemia, despite their greater metabolic importance in the immature heart in determining ischemic tolerance [4]. An understanding of the baseline metabolic profile before the stresses of cardioplegic arrest are scrutinized would seem to be a prerequisite for developing a coherent and rational approach to myocardial protection.
Little is also known about the effects of cyanosis and age on AA levels. Experimental models have suggested that acute hypoxia depletes endogenous AAs but there are few data on the effects of chronic hypoxia, partly due to the difficulties in establishing a chronically hypoxic animal model [7, 8]. We have recently demonstrated in pediatric patients undergoing open-heart surgery that cyanosis is associated with higher lactate concentrations and young age with higher adenine nucleotide levels [9], which may explain the observation that cyanotic hearts appear more vulnerable to ischemia-reperfusion injury than acyanotic hearts [5, 6]. The pathology affecting the heart also has important implications for AA metabolism, with ischemic adult hearts having higher concentrations of alanine and branched chain amino acids (BCAA) than hypertrophic hearts of patients with aortic stenosis [10]. This finding implies important differences in energy metabolism and protein turnover attributable to pathology. However, in pediatric hearts there are few important differences in adenine nucleotide levels attributable to pathology [9]. Identification of metabolically stressed subsets of patients would be useful in tailoring cardioplegic solutions to the individual rather than uncritically applying one technique to all patients.
The aim of this study was to describe the preischemic AA profile of the immature pediatric heart with congenital heart disease by measuring intracellular free AA concentrations and to investigate the effects of cyanosis, age, and pathology on this. This work on amino acids represents the second part of a larger study comprehensively examining the baseline metabolic profile of the pediatric heart with congenital heart disease; the first part, which assessed adenine nucleotide levels in the same group of patients, has been published previously [9].
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Material and Methods
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Children undergoing elective repair of congenital heart defects over an 18-month period at the Royal Hospital for Children, Bristol, were recruited. The study was approved by the local Institutional Review Board (23 February 1996) and the parents-guardians of all patients gave written informed consent.
Anesthetic and surgical techniques were as reported previously [9]. A transmural right ventricular biopsy (mean weight ± SD, 3.7 ± 2.7 mg) was collected immediately prior to cross-clamping the aorta using an 18G x 6 cm Trucut biopsy needle (Allegiance Healthcare, McGaw, IL). The specimen was immediately frozen in liquid nitrogen; amino acids in the neutralized extract were separated and quantified by use of high-performance liquid chromatography. Data for the concentrations of the following amino acids were analyzed for this study: aspartate, glutamate, glutamine, taurine, alanine, and the branched chain amino acids (namely leucine, isoleucine, and valine). The alanine to glutamate ratio (AGR), a marker of metabolic stress, was also calculated [10]. Protein determination was carried out according to the Lowry method using a protein determination kit from Sigma Diagnostics (Poole, Dorset, UK) with bovine serum albumin as a standard. Amino acid concentrations were calculated as nmol per mg protein content.
Statistical Analysis
Descriptive demographic and clinical data were summarized using medians and interquartile ranges or frequencies. Comparative analyses were carried out to estimate the effects of cyanosis, age, and diagnosis on amino acid concentrations, as far as possible taking into account likely confounding factors. Because the raw data were positively skewed, data describing the concentrations of amino acids and AGR were transformed by taking natural logarithms to normalize their distributions. Effects are therefore expressed as relative changes in geometric means.
Two analytical approaches were used to estimate the effect of cyanosis (defined as a resting arterial oxygen saturation [SaO
2] < 90%). First, age-matched pairs of acyanotic and cyanotic children 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. Acyanotic children had to have a diagnosis of ventricular septal defect (VSD) and cyanotic children diagnoses of either tetralogy of Fallot (TOF) or pulmonary atresia with VSD (PA+VSD) to control as much as possible for any confounding effects of specific pathologies. Paired t tests were used to estimate the effects of cyanosis. Second, multiple regression modeling was used to analyze the data for all children aged between 1 month and 4 years. The cutoff upper age was selected prior to analysis, based on the age distribution among cyanotic and acyanotic children; very few children over 4 years were cyanotic. A third analysis estimated the effects of age and pathology in acyanotic children only, also by multiple regression modeling.
Regression models were fitted interactively. Unadjusted models for cyanosis and age included just these variables. Adjusted models also considered gender, 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 the variables were of primary interest to the analysis.
The number of amino acids of interest necessitated many statistical comparisons. No correction to the criterion for statistical significance (p < 0.05) was made to take into account multiple comparisons. However, the number of comparisons, the consistency of the findings, and their magnitude were all factors in our interpretation as well as their statistical significance. All data were analyzed using STATA version 8.2 (STATA Corporation, College Station, TX).
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Results
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A total of 181 children were recruited (37, 20% cyanotic); 113 were aged 4 years or less and only one was less than 1 month. Cyanotic patients, who all had either TOF or PA+VSD, were younger and had lower SaO
2 and higher Hb concentrations than acyanotic patients (Table 1). The protein content of myocardial tissue was similar in cyanotic and acyanotic hearts (mean, 9.9% vs 9.62% of wet weight, respectively; difference between means = 0.33%, 95% CI 1.23 to 1.90, p= 0.68). The metabolic data for amino acids are summarized in Table 2
in natural units.
Effects of Cyanosis
Because of differences in age, only 22 pairs of cyanotic (TOF or PA+VSD) and acyanotic children (VSD) could be age-matched. The mean differences between the 22 pairs of children were not significant for any of the six amino acids analyzed or for AGR (p > 0.15). Adjusted and unadjusted estimates of the effects of 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+VSD (cyanotic) in order to control as much as possible for the effect of diagnosis (n = 74). There was little evidence of an effect of cyanosis on amino acid concentrations or AGR other than for alanine, the estimate of which from both unadjusted and adjusted regression analyses suggested that levels were about 20% higher in cyanotic than acyanotic children (p = 0.04).
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Table 3. Unadjusted and Adjusted Estimates of the Effect of Cyanosis on Amino Acid Concentrations in Children Aged 1 Month to 4 Years
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Effects of Age and Pathology in Acyanotic Hearts
The effects of age on amino acid levels in acyanotic patients are complex (Table 4
and Fig 1). The concentrations of glutamate, aspartate, taurine, alanine, and the BCAAs were all significantly associated with age. After controlling for diagnosis, concentrations of glutamate, aspartate, and alanine decreased from birth to about 8 to 10 years, then started to increase again (p < 0.05 for both linear and quadratic terms); concentrations of taurine and BCAAs decreased steadily with increasing age (p < 0.05); there was no association at all between age and AGR (p = 0.97). Glutamine also declined with age but, after adjustment for other factors, this change was not significant.
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Table 4. Unadjusted and Adjusted Estimates of the Effect of Increasing Age on Amino Acid Concentrations in Acyanotic Children
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Fig 1. Relationships of amino acid concentrations to age in acyanotic children. Fitted lines represent the regression models described in Table 4 for children with ventricular septal defect (lines for other pathologies would have exactly the same shape but be translated up or down in accordance with the results shown in Table 5).
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The effects of different acyanotic pathologies on the basal metabolic state were expressed in relation to children with VSDs (the largest diagnostic group, n = 48, Table 5). Significance tests of the variance in amino acid levels explained by diagnosis were significant for glutamate (p = 0.006), glutamine (p = 0.003), and BCAAs (p = 0.004). Patients with ASDs (n = 43) had higher levels of glutamate but lower concentrations of glutamine and branched chain AAs compared with patients with VSDs. Acyanotic patients with TOF (n = 11) also had higher concentrations of glutamate and tended (p = 0.07) to have higher concentrations of glutamine; their BCAA concentrations were very similar to those of patients with VSDs. Concentrations of AAs in patients with other acyanotic pathologies (n = 42) did not differ significantly from concentrations in patients with VSDs.
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Table 5. Adjusted Estimates of the Effects of Diagnosis, Compared With VSD (n=48), on Amino Acid Concentrations in Acyanotic Children
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Comment
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This study demonstrates that there was little effect of cyanosis on the myocardial concentrations of any amino acid other than alanine, which was approximately 20% higher in cyanotic patients. In acyanotic patients the relationships between amino acid concentration and age were complex; the concentrations of glutamate, aspartate, and alanine decreased from birth to about 8 to 10 years then started to increase, whereas the concentrations of taurine and BCAAs decreased steadily with increasing age. Glutamate, glutamine, and BCAA levels were all associated with pathology; patients with VSDs (and hence a greater volume-pressure load) had evidence of greater metabolic stress (lower glutamate levels, higher BCAA levels) compared with patients with ASDs.
Study Limitations
The concentrations of amino acids, particularly in cyanotic hearts, may have been affected by two factors. First, variation in 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. Second, an unintended reoxygenation injury at the induction of anesthesia and at the commencement of CPB, where the priming fluid is hyperoxic. Although acyanotic hearts will undergo the same injury, this has been demonstrated to occur earlier and be greater in cyanotic hearts [11].
This study included only one neonate and thus additional studies would be necessary to determine if these findings can be extrapolated to younger patients. It also had limited power to detect a metabolic difference 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.
Myocardial Amino Acid Metabolism
Cardiac muscle is a metabolic omnivore, which can derive its energy from a variety of fuels, including fatty acids (primary source for mature myocardium), glucose and lactate (primary source for immature myocardium), ketone bodies, and free amino acids. The greatest constituents of the free AA pool are the nonessential amino acids, including glutamate and aspartate, which play a vital role in the malate-aspartate shuttle, essential for balancing reducing equivalents between the cytoplasm and mitochondria [12]. As anaplerotic agents all the nonessential amino acids can supplement into the Krebs cycle, which helps to counteract disturbances in Krebs cycle activity such as those that occur during loading with glucose, acetate, ketone bodies, or fatty acids or under pathologic conditions including aerobic arrest and ischemia [13]. Recent work has also suggested that the cardioprotective effect of glutamate may be mediated through an enhanced ability to destroy cellular reactive oxygen species by stimulation of glutathione peroxidase [14]. Both glutamate and aspartate are used during hypoxia and ischemia to generate energy by transamination and substrate level phosphorylation allowing production of 2 mol ATP over that formed by anaerobic lactate catabolism [3, 15, 16].
Cyanosis
Although abundant data are available concerning the effects of hypoxia on the adult myocardium, very little is known about the influence of oxygen deprivation on developing cardiac muscle. Experimental models have demonstrated that acute hypoxia depletes myocardial glutamate, aspartate [7], and taurine [17]. However, acute preparations do not allow for the development of the adaptive mechanisms that accompany chronic hypoxia so their results must be interpreted in this context. This is clearly demonstrated by our data and that of Imura and colleagues [6], which showed no effect of cyanosis on glutamate, aspartate, or taurine levels. However, alanine concentrations were about 20% higher in cyanotic hearts, which is consistent with the greater use of glutamate as a source of energy during hypoxia with the formation of alanine as a by-product [18]. The relationship between hypoxic stress and alanine is confirmed by the finding that alanine positively correlated with BCAA and lactate (p < 0.0001, data not shown). The AGR, which has been demonstrated to be a marker of metabolic stress in adult hearts [10], tended to be higher in cyanotic patients (by 16%) although this did not reach statistical significance.
Age
Our data are consistent with previous experimental and clinical work demonstrating higher levels of glutamate and aspartate in immature canine hearts (6–8 weeks old) compared with adult hearts [3], and in acyanotic infants compared with acyanotic children [6]. Similarly, in rat hearts taurine declines with age with a strong negative correlation between taurine content and resistance to an ischemic insult [19]. This is consistent with recent work demonstrating infant hearts to be more vulnerable to ischemia than those of children [6, 20]. Low levels of taurine may protect against ischemic injury by reducing the osmotic load and regulating [Na+]i by the Na+ / taurine symport [21]. It is beyond the scope of this work to provide a mechanism for these age-related changes in AA levels, but either greater intrinsic production-extrinsic supply of AAs or reduced catabolism seem likely. For example, enzymes associated with fatty acid metabolism, the Krebs cycle, and respiratory chain, together with creatine kinase and various cytochromes, have been shown to have a low activity in the immature heart [22].The reason for the increase in concentrations of glutamate, aspartate, and alanine beyond 8 to 10 years of age is not immediately apparent but is nevertheless in keeping with values previously reported in ischemic and hypertrophic adult hearts [10].
The concentrations of BCAAs also declined with age. Although BCAA levels correlate with metabolic stress, this is unlikely to be the cause as there was no significant effect of age on the alanine to glutamate ratio. The BCAAs, and leucine in particular, are involved in the regulation of myocardial protein turnover [23, 24]. For instance, under different conditions leucine is known to inhibit protein degradation, accelerate protein synthesis, and improve nitrogen balance [25]. During myocardial ischemia protein synthesis and degradation become inhibited as a means of conserving energy. Changes in BCAA levels with age may reflect ontological differences in protein turnover.
The first stage in the metabolism of BCAAs involves reversible transamination to the 2-oxo acid, followed by an irreversible oxidative-decarboxylation step, catalyzed by the branched chain 2-oxo acid dehydrogenase multienzyme complex [26]. The regulation of this enzyme is multifaceted involving both hormonal (glucocorticoids) and phosphorylation (protein kinases) controls [27]. Zhao and colleagues [28] demonstrated that the activity of this enzyme complex increases as a function of age in neonatal rat hearts, with only about 30% to 45% of the enzyme being active throughout the suckling period. This may explain the change in myocardial BCAA levels demonstrated in our work.
Pathology
Many infants with congenital heart disease are exposed to volume and pressure loading secondary to a large left-to-right shunt or ventricular outflow obstruction, which may result in unfavorable metabolic changes in the myocardium [29]. This is demonstrated in our data, where patients with VSDs (and hence a greater volume-pressure load) had evidence of greater metabolic stress with lower levels of glutamate, higher concentrations of BCAAs, and a trend to higher AGRs compared with patients with ASDs. Our previous work reflects this with a trend toward lower ATP/ADP ratios and higher lactate concentrations in patients with VSDs [9]. Thus far, little account has been taken of the differing volume-pressure loading characteristics of the ventricle and the effect on myocardial protection, but it is reasonable to suggest that those immature hearts with a greater volume-pressure load, which are therefore relatively depleted of important AAs used as anaerobic energy sources, would be the ones to benefit most from techniques that have been shown to resuscitate metabolically stressed hearts (eg, blood cardioplegia, amino acid enrichment) [30].
In summary, this study demonstrates that age is an important factor determining the baseline amino acid profile of the pediatric heart with young age being associated with higher levels of aspartate, glutamate, taurine, alanine, and branched chain AAs. Chronic hypoxia leads to increased levels of alanine but does not deplete endogenous AAs, as experimental preparations of acute hypoxia would suggest. There were significant effects of pathology on glutamate, glutamine, and branched chain AAs with evidence of greater metabolic stress in patients with VSDs (and hence greater volume-pressure loading) compared with ASDs.
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Acknowledgments
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We would like to thank Dr Nicola King, PhD, for suggestions during manuscript preparation and Anne Moffat, Svitlana Korolchuk, and Mark Ginty for expert technical assistance. This work was funded by The British Heart Foundation and The National Heart Research Fund.
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Abstract
Introduction
