**4.1 Characteristics of methodology on myocardial energy metabolism study**

In humans, the coronary sinus, which empties into the right atrium, receives blood from 96% of veins from the left ventricular free wall and septum (Sethna et al., 1986). The coronary sinus system drains approximately three fourths of the blood entering the left coronary artery and only 10 to 20 % of the inflow of the right coronary artery. The rate of tissue metabolism (uptake or release) can only be measured by multiplying the arterycoronary vein difference by the blood flow if the flow, the arterial concentration, and the rate of tissue metabolism are all constant. We did not measure coronary sinus blood flow in this study because of technical difficulties for infants. Then, we calculated oxygen extraction ratio for standardizing and comparing the substrate use in the heart.

This kind of studies to adult patients carried without heparinization but with frequent wash of catheter for prevention of thrombus formation, since it is well known that heparin induces the production of free fatty acids from lipoprotein by activation of lipoprotein lipase. We used, in this study, heparin for anti-coagulation and obtained blood samples

infusion of low dose lactate and glucose did not influence the concentrations of both lactate

We calculated myocardial OER of each substrate since, in this study, coronary sinus blood flow could not be measured. Figure 1 shows OER of each substrate in each group. Glucose OER in each patient was quite variable so that there was no significant difference on the mean value; 2.0±13.0% for KD group, 8.4±11.0% for ASD group, and 15.5±20.4% for PH group. Mean arterio-venous difference of lactate in PH group was negative resulting in - 5.3±11.2% of calculated lactate OER. This value was significantly lower than both of KD group (7.8±9.2 %, p=0.013) and of ASD group (19.7±9.5, p=0.004). On the other hand, the lactate OER of ASD group showed higher trend than both KD group and PH group. There were no significant difference on FFA OER in each group; 62.8±28.2% for KD group, 63.6±9.8% for ASD group, and 62.8±28.0% for PH group. Sum of each glucose, lactate, and

The lactate/pyruvate (L/P) ratios in coronary vein were similar among the groups. However, the L/P ratios of both ASD group and PH group were relatively higher values than those of KD group. Each values of redox potential (Eh) calculated from blood lactate and pyruvate showed no significant difference among groups. The ΔEh also showed no significant difference among the groups but the ΔEh of PH group was relatively lower value

As some patients in PH group were supposed myocardial relative ischemia or hypoxic state, we measured the major energy substrates under administration of oxygen for CHD patients. Figure 2 demonstrates the change of lactate OER both from ASD group and PH group. Lactate OER of ASD group did not change with oxygen inhalation. On the other hand, its PH group increased from -6.3±10.9% to 3.0±9.9%. However, of interest, both the CS L/P ratio and ΔEh of each group showed no remarkable changes even after inhale of oxygen

**4.1 Characteristics of methodology on myocardial energy metabolism study** 

ratio for standardizing and comparing the substrate use in the heart.

In humans, the coronary sinus, which empties into the right atrium, receives blood from 96% of veins from the left ventricular free wall and septum (Sethna et al., 1986). The coronary sinus system drains approximately three fourths of the blood entering the left coronary artery and only 10 to 20 % of the inflow of the right coronary artery. The rate of tissue metabolism (uptake or release) can only be measured by multiplying the arterycoronary vein difference by the blood flow if the flow, the arterial concentration, and the rate of tissue metabolism are all constant. We did not measure coronary sinus blood flow in this study because of technical difficulties for infants. Then, we calculated oxygen extraction

This kind of studies to adult patients carried without heparinization but with frequent wash of catheter for prevention of thrombus formation, since it is well known that heparin induces the production of free fatty acids from lipoprotein by activation of lipoprotein lipase. We used, in this study, heparin for anti-coagulation and obtained blood samples

and glucose since blood levels of those substrates were within the normal values.

FFA OER was calculated as a total OER of heart.

**3.5 The effects of oxygen inhalation** 

than other groups.

(Table 3).

**4. Discussion** 

**3.4 Myocardial redox state or anaerobic metabolism (Table 3)** 

under heprinized state because of two reasons; 1) for preservation of veins and arteries from obstruction in younger children and 2) for our aim of studying myocardial metabolism in patients under critical states as in pediatric intensive care unit or in surgical intervention where many patients were heparinized.

In spite of these limitation, this method we applied here is still useful for clinical study on myocardial metabolism (Vánky et al. 2006) because data obtained are supposed not far from animal model study (Lopaschuk et al., 1992), computer simulation study and isotopical study in human.

#### **4.2 Myocardial use of lactate and other substrates in non-cyanotic CHD**

It is very important to know the myocardial energy substrate use *during the management of heart failure* or cardiac surgery of children with CHD. However, myocardial metabolism even in the normal immature heart has not been fully elucidated. Although data we can refer on myocardial energy substrate use in normal children are limited, myocardial fatty acids uptake of KD group *resembles the results that Rudolph demonstrated* (Rudorph et al., 1971). For this reason, we considered that results from KD group represented normal myocardial substrate use in children. Table 4 shows the comparison among some previous reports on the substrates use in hearts in young including cyanotic CHD. Myocardial FFA uptake in children shows very similar levels among the reports. The very variable glucose uptake shown in other reports including adults suggested that glucose may not play an important role for myocardial energy supply for children at rest. (Vánky et al. 2006,Lopaschuk et al., 1992).

It has been demonstrated that adult hypertrophied hearts prefer to oxidize glucose. Increase of glucose oxidation may be beneficial for hypertrophied heart on production of ATP with less myocardial oxygen consumption than fatty acid oxidation. Allard *et al* reported that the steady-state palmitate oxidation rates were decreased in the hypertrophied hearts compared with control hearts (Allard et al., 1994). Although the uptake of glucose of CHD hearts, in our study, was quite variable, both hearts with the volume overloaded RV (ASD group) and with the pressure overloaded RV (PH group) showed tendency of increase of glucose uptake. (Figure 1, Table 4). These suggest that a myocardial potential of glucose use in children with CHD may not be an inferior level in comparison with adult hearts against overload. However, *one should note in our results that* FFA use was high levels even in PH group and that lactate was dominant energy supplier more than glucose in ASD group.

Gertz *et al* have reported that in subjects with high blood free fatty acids, myocardial lactate extraction may be low (Gertz et al., 1980). However, this is not the case at least in children with CHD (Table 4). The lactate use including of cyanotic CHD is relatively high even under the high levels of fatty acid use. From another point of view, it is speculated that fatty acid use in children with CHD *have reached to* near maximum levels and, as a result, lactate regulated the energy supply against additional loads on the heart. Some studies have clarified that fatty acids oxidation increased with elevation of ventricular workload in immature hearts (Itoi et al., 1993a,Ascuitto et al., 1999). The lactate oxidation rates of the immature hearts were also increased by the addition of preload to the RV without significant change of glucose oxidation (Itoi et al., 1993b). The ASD group in our study showed the very same result of this experimental model on change of the lactate oxidation (Figure 1). Recently, Vánky *et al* revealed that no significant uptake of glucose was detected before or after surgery for aortic stenosis but the uptake of lactate was significant before surgery (Vánky et al. 2006).

Myocardial Lactate Metabolism in Children with Non-Cyanotic Congenital Heart Disease 325

Our results suggested that, under the high potential of fatty acids oxidation, 1) the low level of acceleration of oxidative metabolism as in ASD group resulted in increasing of lactate oxidation for filling NADH because of limitation of glycolysis activity by fatty acids oxidation, 2) the higher level of cardiac work as in PH group results in the faster rates of glycolysis by cellular hypoxia and/or adrenaline (Brooks et al., 2002, Massie et al., 1995), which were also accompanied by increased conversion of pyruvate to lactate by over-

OER Glucose

KD ASD PH

0

20

40

60

80

100

120

KD ASD PH

OER FFA

KD, Kawasaki disease; ASD, atrial septal defect; PH, pulmonary hypertension; \*, significantly different from KD; \*\*, significantly different from ASD.

KD ASD PH



0

20

40


OER Lactate


0

10

20

30

40

50

Fig. 1. Myocardial oxygen extraction rate (OER) of glucose, lactate, and fatty acids

\*,\*\*

production of NADH (Figure 3).

The blood lactate levels in the resting state are low, in the range of 0.5-1 mM, in human adults. Results of our study showed that, even in children with CHD, arterial lactate levels were the same as in adults (Table 2). Then, the lactate use changes in hearts of children with non-cyanotic CHD might not be influenced by blood lactate levels. Lactate oxidation occurs because the lactate dehydrogenase (LDH) isozyme found in heart has a low affinity for pyruvate, although the equilibrium constant for the LDH is in the direction of lactate formation. In addition, the hydrogen ion, pyruvate, and NADH formed by the LDH reaction are rapidly removed in the aerobic heart, forcing the reaction in the direction of the formation of pyruvate (Drake-Holland, 1983). Furthermore, in the setting of a fully activated FFA oxidation, glycolysis flux and the pyruvate dehydrogenase complex (PDC) activity are supressed with increased NADH from β-oxidation. This phenomenon may result in not only deceleration of glucose oxidation but also acceleration of lactate oxidation (Figure 3). This scenario may happen in mildly overloaded hearts as in ASD group.

The very characteristic finding in our study was the efflux of lactate under the stable fatty acids use in PH group (Figure 1). In RVH, there is a mitochondrial metabolic switch from glucose oxidation to glycolysis due to myocardial ischemia (Pio et al., 2010, Gomez et al., 2001). Positron emission tomography studies in patients with RVH suggested that there is increased RV glucose uptake, which is thought to reflect enhanced glycolysis. The less efficient production of ATP by glycolysis in RVH meant the formation of lactate, rather than pyruvate (Oikawa et al., 2005). In the immature heart, lactate dehydrogenase (LDH), which is predominated by the M type isozyme, as higher activity, resulting in greater lactate production from pyruvate (Brooks et al., 2002). This requires greater NADH levels than seen in the adult heart. The dominance of glycolytic flux in immature hearts leads to accumulation of lactate to a greater extent than is seen in adult hearts during profoundly hypoxic states (Brooks et al., 2002). Now, does the spillover of lactate from hearts of PH group indicate the existence of profound myocardial ischemia of the right ventricle?

#### **4.3 Redox-potential of the lactate-pyruvate system in CHD**

The redox-potential of the coronary sinus blood approaches that of cardiac tissues, and the redox-potential of the coronary venous blood becomes more negative than that of arterial blood. When ΔEh is positive there is active cellular oxidation and the energy required is supplied by oxidative phosphorylation. When ΔEh is negative there is glycolysis and anaerobic phosphorylation becomes an important energy source (Gudbjarnason & Bing, 1962). The RV overloaded heart, especially PH group, showed a tendency of decrease of ΔEh (Table 3). Since some hearts of CHD were supposed to be under hypoxic state, we administered oxygen to patients. The results that oxygen inhalation increased influx of lactate (Figure 2) without changes of both the L/P ratio and ΔEh (Table 3) suggested that myocardial hypoxic state may not be only one cause of the lactate efflux from hearts of the PH group.

Kobayashi *et al* demonstrated that, in isolated perfused heart, both the intracellular and the perfusate L/P ratio increases at higher cardiac workloads (Kobayashi & Neely, 1979). The L/P ratio of a given cell is thought to reflect the cytosolic NADH/NAD ratio (Rassmussen et al., 2009). Since the coronary venous L/P ratio at rest has been reported around 10 (Friedli 1977), our results suggested that the cytosolic NADH/NAD ratio may be higher in the CHD groups, although statistically not significant, than in KD group at rest (Table 3).

The blood lactate levels in the resting state are low, in the range of 0.5-1 mM, in human adults. Results of our study showed that, even in children with CHD, arterial lactate levels were the same as in adults (Table 2). Then, the lactate use changes in hearts of children with non-cyanotic CHD might not be influenced by blood lactate levels. Lactate oxidation occurs because the lactate dehydrogenase (LDH) isozyme found in heart has a low affinity for pyruvate, although the equilibrium constant for the LDH is in the direction of lactate formation. In addition, the hydrogen ion, pyruvate, and NADH formed by the LDH reaction are rapidly removed in the aerobic heart, forcing the reaction in the direction of the formation of pyruvate (Drake-Holland, 1983). Furthermore, in the setting of a fully activated FFA oxidation, glycolysis flux and the pyruvate dehydrogenase complex (PDC) activity are supressed with increased NADH from β-oxidation. This phenomenon may result in not only deceleration of glucose oxidation but also acceleration of lactate oxidation (Figure 3). This

The very characteristic finding in our study was the efflux of lactate under the stable fatty acids use in PH group (Figure 1). In RVH, there is a mitochondrial metabolic switch from glucose oxidation to glycolysis due to myocardial ischemia (Pio et al., 2010, Gomez et al., 2001). Positron emission tomography studies in patients with RVH suggested that there is increased RV glucose uptake, which is thought to reflect enhanced glycolysis. The less efficient production of ATP by glycolysis in RVH meant the formation of lactate, rather than pyruvate (Oikawa et al., 2005). In the immature heart, lactate dehydrogenase (LDH), which is predominated by the M type isozyme, as higher activity, resulting in greater lactate production from pyruvate (Brooks et al., 2002). This requires greater NADH levels than seen in the adult heart. The dominance of glycolytic flux in immature hearts leads to accumulation of lactate to a greater extent than is seen in adult hearts during profoundly hypoxic states (Brooks et al., 2002). Now, does the spillover of lactate from hearts of PH

group indicate the existence of profound myocardial ischemia of the right ventricle?

The redox-potential of the coronary sinus blood approaches that of cardiac tissues, and the redox-potential of the coronary venous blood becomes more negative than that of arterial blood. When ΔEh is positive there is active cellular oxidation and the energy required is supplied by oxidative phosphorylation. When ΔEh is negative there is glycolysis and anaerobic phosphorylation becomes an important energy source (Gudbjarnason & Bing, 1962). The RV overloaded heart, especially PH group, showed a tendency of decrease of ΔEh (Table 3). Since some hearts of CHD were supposed to be under hypoxic state, we administered oxygen to patients. The results that oxygen inhalation increased influx of lactate (Figure 2) without changes of both the L/P ratio and ΔEh (Table 3) suggested that myocardial hypoxic state may not be only one cause of the lactate efflux from hearts of the

Kobayashi *et al* demonstrated that, in isolated perfused heart, both the intracellular and the perfusate L/P ratio increases at higher cardiac workloads (Kobayashi & Neely, 1979). The L/P ratio of a given cell is thought to reflect the cytosolic NADH/NAD ratio (Rassmussen et al., 2009). Since the coronary venous L/P ratio at rest has been reported around 10 (Friedli 1977), our results suggested that the cytosolic NADH/NAD ratio may be higher in the CHD

groups, although statistically not significant, than in KD group at rest (Table 3).

scenario may happen in mildly overloaded hearts as in ASD group.

**4.3 Redox-potential of the lactate-pyruvate system in CHD** 

PH group.

Our results suggested that, under the high potential of fatty acids oxidation, 1) the low level of acceleration of oxidative metabolism as in ASD group resulted in increasing of lactate oxidation for filling NADH because of limitation of glycolysis activity by fatty acids oxidation, 2) the higher level of cardiac work as in PH group results in the faster rates of glycolysis by cellular hypoxia and/or adrenaline (Brooks et al., 2002, Massie et al., 1995), which were also accompanied by increased conversion of pyruvate to lactate by overproduction of NADH (Figure 3).

KD, Kawasaki disease; ASD, atrial septal defect; PH, pulmonary hypertension; \*, significantly different from KD; \*\*, significantly different from ASD.

Fig. 1. Myocardial oxygen extraction rate (OER) of glucose, lactate, and fatty acids

Myocardial Lactate Metabolism in Children with Non-Cyanotic Congenital Heart Disease 327

KD ASD PH ANOVA *P*

Age (year) 3.5±2.3 6.9±3.0 2.1±0.8† 0.005 HR (bpm) 123±24 118±1 129±27 NS RVPsys (mmHg) 24±4 40±12\* 80±22\*\*, † <0.001 LVPsys (mmHg) 112±15 116±9 100±10 NS RVP/LVP 0.22±0.05 0.35±0.13 0.79±0.17\*\*,† <0.001 LV DP (x1000) 13.63±2.06 13.61±1.7 12.95±2.82 NS RV DP (x1000) 2.94±0.65 4.80±1.94 10.32±3.28\*\*,† 0.001

Qp/Qs 1 1.8±0.2 1.7±0.5 - Hb(g/dl) 12.5±0.7 13.3±0.7 12.9±0.9 NS

ratio; Hb, hemoglobin

Aorta

Coronary sinus

Table 1. Patients profiles

significantly different between ASD vs VSD

Table 2. Myocardial substrate uptake

HR, heart rate; RVPsys, systolic right ventricular pressure; LVPsys, systolic left ventricular pressure; DP, double products (=ventricular systolic pressure x heart rate); Qp/Qs, pulmonary-sysytolic flow

\*, significantly different between KD vs ASD; \*\*, significantly different between KD vs VSD; †,

KD ASD PH ANOVA *p* 

O2 sat (%) 97.7±0.5 97.1±0.8 95.1±3.3 NS Glucose (mmol/L) 4.95±1.07 5.48±0.33 5.17±0.88 NS lactate (mmol/L) 0.72±0.19 0.83±0.44 0.86±0.36 NS pyruvate (mmol/L) 0.045±0.023 0.048±0.015 0.092±0.093 NS FFA (mmol/L) 1.28±0.33 1.41±0.44 1.34±0.3 NS

O2 sat (%) 31.5±4.3 35.2±12.5 32.2±5.6 NS Glucose (mmol/L) 4.93±1.07 5.46±0.28 5.18±0.95 NS lactate (mmol/L) 0.53±0.16 0.53±0.3 0.95±0.47\*\*,† 0.033 pyruvate (mmol/L) 0.052±0.027 0.037±0.01 0.1±0.081 NS FFA (mmol/L) 1.08±0.34 1.2±0.46 1.15±0.3 NS

\*\*, significantly different from ASD group; †, significantly different from ASD group

ASD, atrial septal defect; PH, pulmonary hypertension. \*, significantly different from ASD; \*\*, significantly different from oxygen -

Fig. 2. Effects of oxygen administration of myocardial lactate use

LDH, lactate dehydrogenase; PDC, pyruvate dehydrogenase complex.

NADH produced by glycolysis or conversion of lactate to pyruvate is carried into the mitochondrial matrix via NADH shuttle. In mitochondrial matrix, NADH is produced from conversion of pyruvate to acetyl-CoA catalyzed by PDC.

Fig. 3. Relationship between myocardial energy substrate use and pathways for oxidation of NADH


HR, heart rate; RVPsys, systolic right ventricular pressure; LVPsys, systolic left ventricular pressure; DP, double products (=ventricular systolic pressure x heart rate); Qp/Qs, pulmonary-sysytolic flow ratio; Hb, hemoglobin

\*, significantly different between KD vs ASD; \*\*, significantly different between KD vs VSD; †, significantly different between ASD vs VSD

Table 1. Patients profiles

326 Congenital Heart Disease – Selected Aspects

ASD, atrial septal defect; PH, pulmonary hypertension.

\*, significantly different from ASD; \*\*, significantly different from oxygen - Fig. 2. Effects of oxygen administration of myocardial lactate use

LDH, lactate dehydrogenase; PDC, pyruvate dehydrogenase complex.

acetyl-CoA catalyzed by PDC.

NADH

NADH produced by glycolysis or conversion of lactate to pyruvate is carried into the mitochondrial matrix via NADH shuttle. In mitochondrial matrix, NADH is produced from conversion of pyruvate to

Fig. 3. Relationship between myocardial energy substrate use and pathways for oxidation of


\*\*, significantly different from ASD group; †, significantly different from ASD group Table 2. Myocardial substrate uptake

Myocardial Lactate Metabolism in Children with Non-Cyanotic Congenital Heart Disease 329

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CV, coronary vein; Eh, redox potential, ΔEh, difference of redox potential between artery and coronary vein

Table 3. Anaerobic Metabolism
