We demonstrated the superiority of Custodiol-N above Custodiol in terms of cardiac and endothelial function after CPB in our clinically relevant canine model of cardiopulmonary bypass.
Since the start of cardiac surgery in the 1950s, multiple techniques (hypothermia, crystalloid or blood cardioplegic solutions, direction of introduction (antegrade or retrograde) etc.) have been used to protect the heart during the surgical requirement for elective global ischemia. However, no single method was unequivocally the best. There is a clinical and experimental evidence of IR injury after CPB. Even if cardiac dysfunction is not always clinically remarkable after CPB, reduction of myocardial contractility may occur as described in a human study using pressure–volume relationships . We could also show in our previous canine studies, that myocardial dysfunction was developed after the administration of Custodiol using a sensitive pressure-volume conductance catheter .
The new cardioplegic solution (Custodiol-N) was modified in 4 important points to enhance the protective capacity of the Custodiol solution: 1, the addition of iron-chelators (desferoxamine and the new, membrane-permeable LK-614) to reduce iron-dependent injury 2, reduction of chloride concentration to reduce chloride-induced injury 3, addition of cardioprotective amino acids (L-Arginin: NO-precursor to improve coronary blood flow; glycine and alanine to stabilize the plasma membrane) 4, partially substitution of histidine by N-α-acetyl-L-histidine to inhibit the histidine-induced cytotoxicity.
The combination of hypothermia and the potassium-induced cardiac arrest represent the basis concept of cold crystalloid cardioplegic solutions. However, present studies clearly demonstrated that cold storage activates specific cardiac injury processes; production of ROS triggered by a cold-induced rise of physiologically negligible cellular pool of the cellular cheletable iron, which inhibit strongly cardiac and endothelial function since redox-active iron ions convert ROS of low toxicity into highly reactive species such as hydroxyl radicals [17,18]. Rauen and co-workers determined the chelatable iron pool in cultured rat hepatocytes and showed that at low temperature the chelatable iron pool rapidly increased and remained elevated for some hours . In a further investigation, Rauen et al. demonstrated the effectiveness of a non-permeable iron-chelator deferoxamine and a new membrane permeable iron-chelator LK-614 against iron-dependent cold injury .
Recently, a new, iron-independent component of cold induced injury has also been described. When the stronger iron-dependent component was inhibited with iron chelators, the previously overshadowed chloride-dependent injury became the limiting factor of cell survival. The new solution (Custodiol-N) contains only 30 mM of chloride because this concentration was sufficient to inhibit the iron-independent component of cold induced injury in cultured rat hepatocyte; there was no need to eliminate chloride completely from the medium .
An experimental study clearly showed that application of L-arginine as a substrate of eNOS can improve postischemic blood flow and decrease leukocyte adhesion . Moreover, a recent human study described that application of L-arginin into the cardioplegic solution improved left ventricular diastolic function . Two amino acids, alanine and glycine, are also included in Custodiol-N in order to decrease the sodium accumulation during cold ischemia and to stabilize the plasma membrane .
Side effects of using Custodiol solution were previously also described (8). The main component of Custodiol, histidine, is an excellent physiological buffer. However, Rauen et al. demonstrated that histidine has a potential negative effect on cell survival during cold ischemia (called as preservation solution toxicity) . With the use of iron chelators (deferoxamine and LK-614), histidine toxicity was strongly inhibited [8,13,22]. They also found that a histidine-derivative, N-acetylhistidine exerted almost no toxicity [8,13]. To prevent the injury caused by histidine, N-acetylhistidine was added into the new Custodiol-N. Radovits et al.  reported in an in vitro experiment a significantly better endothelium protection with an N-acetylhistidine containing solution (without any additives) when compared to Custodiol.
The new cardioplegic solution (Custodiol-N) has already been showed largely improved liver, lung and heart preservation in different experimental studies [9-11,14]. Based on the results of these studies we have tested the effect of Custodiol-N on cardiac and endothelial function in a clinically relevant large animal model. In this study, we investigated global myocardial performance and endothelial function and metabolic parameters of the heart after a 60 minutes cardioplegic arrest. The clinically remarkable parameters such as CO and MAP were unchanged after CPB in both groups. However, we could demonstrate that Custodiol-N is more effective than Custodiol at improving the sensitive, load-independent index of left ventricular contractility (ESPVR, PRSW) after hypothermic cardioplegic arrest and reperfusion. In particular, the PRSW of the left ventricle in the Custodiol-N group remained practically unchanged when compared with the baseline to 60-minute reperfusion values (Figure 1).
To prove whether the Custodiol-N reduce ROS-generation, we also measured biochemical parameter of heart such as plasma myeloperoxidase and plasma nitrite/nitrate level. The increased level of nitrite may be explained as a result of better cardioprotection. Plasma nitrite in blood has been widely used as an index of endothelial NO synthase activity as routine indirect measures of NO levels . Myeloperoxidase (MPO) is mainly released by neutrophil granulocytes, characterised by powerful pro-oxidative properties . MPO catalyzes the conversion of chloride and hydrogen peroxide to hypochlorite and is secreted during inflammatory response. As hypochlorite is extremely toxic to mammalian cells, it causes tissue degradation and DNA breakage. In addition, MPO consumes endothelial-derived NO, thereby reducing NO bioavailability and impairing its vasodilating and anti-inflammatory properties. As shown by the metabolic values, application of new iron-chelators and N-acetyl-L-histidine into Custodiol-N seems to represent an optimal cardioprotection in compared to Custodiol, because hearts of this group showed the best recovery of energy charge potential and cardiac performance. In addition, the increased ATP level in the Custodiol-N group is also a sign of a better cardioprotection.
Cardioplegic solutions were originally designed to protect the myocardium, however at that time the protection of the vasculature, and more importantly, the endothelium, was not an objective. Recently, Radovits et al.  demonstrated the inability of Custodiol solution to offer a reasonable protection and preservation of endothelial function after long-term ischemic storage. Despite the importance of coronary endothelium few investigators examined the effect of Custodiol on endothelial function . In the present study we measured in vivo coronary blood flow before and after 60-min reperfusion. After 60-min reperfusion the basal CBF remained unchanged in the Custodiol-N group while in the Custodiol group decreased significantly when compared to the baseline level (Figure 2).