COMPARISON OF CUSTODIOL AND MODIFIED ST.THOMAS CARDIOPLEGIA FOR MYOCARDIAL PROTECTION IN CORONARY ARTERY BYPASS GRAFTING POREĐENJE KUSTODIOLA I MODIFIKOVANE ST. THOMAS

Background/Aim. Custodiol? is a hyperpolarizing cardioplegic solution which has been used in our national cardiac surgical practice exclusively for the heart transplant surgery. Owing to its numerous advantages over the standard depolarizing solutions, Custodiol? became cardioplegic solution of choice for all other cardiac surgical procedures in many cardio-surgical centers. This study evaluated myocardial protection by Custodiol? compared to modified St. Thomas cardioplegic solution in coronary artery bypass surgery. Methods. In a prospective four-month study, 110 consecutive adult patients who underwent primary isolated elective on-pump coronary artery bypass grafting (CABG) were randomized into the Custodiol? group (n = 54) and the St. Thomas groupa (n = 50), based on the type of administered cardioplegia; six patients were excluded. Cardiac protection was achieved as antegrade cold crystalloid cardioplegia by one of the solutions. Myocardial preservation was assessed through following outcomes: spontaneous rhythm restoration post cross-clamp, and postpoperative cardiac specific enzymes level, ejection fraction (EF) change, inotropic support, myocardial infarction (MI), atrial fibrillation (AF), and death. Results. Preoperative and intraoperative characteristics of patients in both groups were similar except for a considerably longer cross-clamp time in the Custodiol? group (49.1 ? 19.0 vs. 41.0 ? 12.9 minutes; p = 0.022). The Custodiol? group exhibited a higher rate of return to spontaneous rhythm compared to the St. Thomas group (31.5% vs. 20.0%, respectively; p = 0.267), lower rates of AF (20.4% vs. 28%, respectively; p = 0.496), MI (1.8% vs. 10.0%, respectively; p = 0.075) and inotropic support (9.0% vs. 12.0%, respectively; p = 0.651), albeit not statistically significant. There was an insignificant difference in peak value of troponin I between the Custodiol? and Thee St. Thomas group (5.0 ? 3.92 ?g/L vs. 4.5 ? 3.39 ?g/L, respectively; p = 0.755) and creatine kinase-MB (26.9 ? 15.4 ?g/L vs. 28.5 ? 24.2 ?g/L, respectively; p = 0.646) 6 hours post-surgery. EF reduction was comparable (0.81% vs. 1.26%; p = 0.891). There were no deaths in both groups. Conclusions. Custodiol? and modified St.Thomas cardioplegic solution have comparable cardioprotective effects in CABG surgery. The trends of less frequent MI, AF and ino-tropic support, despite the longer cross-clamp time in the Custodiol? group may suggest that its benefits could be ascertained in a larger study.


Introduction
Cardiac surgery is always accompanied by a certain degree of myocardial damage which is multifactorial and cumulative, but ischemia-reperfusion injury is the major factor inducing intraoperative myocardial damage 1 .Myocardial protective strategies during cardiac surgery aim to diminish ischemia-reperfusion myocardial injury that could cause myocardial infarction (MI), arrhythmias, ventricular dysfunction and low cardiac output syndrome (LCOS).Atrial fibrillation, the need for inotropic support, acute renal injury, prolonged intensive care unit (ICU), and death are the most common consequences of LCOS 1,2 .There are numerous methods of myocardial protection during cardiac surgery, but cardiopulmonary bypass (CPB), hypothermia, and cardioplegic arrest remain the primary protective techniques during open heart surgery 2-4 .Cardioplegia, as a method of myocardial protection, has been in use for almost half a century.Crystalloid cardioplegic solutions could be categorized according to the electrolyte composition in two types: extracellular and intracellular type.Extracellular solutions contain higher concentration of sodium, calcium, and magnesium and produce depolarizing cardiac arrest.Intracellular solutions with a low concentration of sodium and calcium, induce cardiac arrest by hyperpolarization.St.Thomas ® Hospital solution and its modifications are the best known and longest used extracellular crystalloid cardioplegia 2 .Bretschneider histidine-tryptophanketoglutarate solution, known as Custodiol ® (Custodiol HTK, Köhler Chemie GmbH, Bensheim, Germany), is an example of an intracellular crystalloid cardioplegia 4 .Custodiol is an intracellular, hyperpolarized, crystalloid solution commonly used for organ preservation in heart transplant procedure, but its use for myocardial protection in ordinary cardiac surgery has not been established entirely and remains an off-label indication in many countries.Custodiol solution administered in a single-dose provides up to 2-3 hours of myocardial protection, which is an advantage over alternative cardioplegic solutions requiring re-administration every 20-30 minutes.Moreover, cardiac arrest induced by hyperpolarization mimics natural resting state of the heart and minimize metabolic demand decreasing ATP depletion thus improving the conditions of the heart to be reanimated at the end of the procedure.Custodiol contains a low sodium concentration that causes hyperpolarization and prevents edema and a high concentration of histidine with high buffering capacity, effective under hypothermic conditions, which may enhance the efficiency of anaerobic glycolysis.Furthermore, Custodiol contains mannitol, ketoglutarate and tryptophan.Mannitol and histidine as well are free radical scavengers that decrease cellular edema.Ketoglutarate is an intermediate in the Krebs cycle and increases energy production upon reperfusion, whereas tryptophan stabilizes cell membranes 4, 5 .
Custodiol has been used in our national cardiac surgical practice exclusively as an organ preserving solution for the heart transplant surgery, with a special supply permission, since it is not yet registered at Medicines and Medical Devices Agency of Serbia (MMDS).Standard strategy, with modified extracellular St.Thomas cardioplegic solution has been used for decades in adult cardiac surgical patients, and thus, it is registered at MMDS.Due to its numerous advantages over the standard, depolarizing solutions, Custodiol became the first choice solution for all other, non-transplant cardiac surgical procedures, in the most cardiac surgical centers worldwide.Our intention to start a new strategy, with routine use of intracellular Custodiol solution for non-transplant cardiac surgical patients, came from well known advantages of this solution.
There is still no clear evidence nor consensus among cardiac surgeons and anesthesiologists as to which cardioplegia provides the best myocardial protection.The ideal composition and method of using cardioplegic solution are still open questions. 4 The aim of this study is to evaluate myocardial protection of Custodiol compared to modified St. Thomas cardioplegia in adult coronary artery bypass surgery.

Patient population
The prospective randomized study included 110 consecutive adult cardiac patients (pts), between February and June 2018, who underwent primary isolated and elective coronary artery bypass grafting surgery (CABG), in condition of extracorporeal circulation.The study protocol followed the Declaration of Helsinki and was approved by the Institution's Ethical Committee.Informed consent was obtained from each patient.Patients who met the inclusion criteria of the study were randomized into two groups: A) receiving standard modified St.Thomas Cardioplegic Solution (St.Thomas M) according to the Hospital protocol, and B) receiving one bolus (20 ml/kg) of Custodiol ® HTK Solution, during six to eight minutes.Six patients were subsequently excluded from the study after randomization due to: myocardial infarction during anesthesia induction -1 patient, errors in the cardioplegia delivery protocol -3 patients, endarterectomy of coronary artery -1 patient, and left ventricular restoration surgery -1 patient.Inclusion Criteria: adult elective coronary patients with minimun two angiographic graftable target vessels (diameter >2.0 mm with stenosis ≥ 70% , left ventricular ejection fraction ≥ 30%, ECHO verified absence of significant valvular pathology.Exclusion criteria: older than 80 years, myocardial infarction within 30 days of the operation, reoperations, urgent/emergent patients, off-pump CABG, left main stenosis >50%, ongoing myocardial ischemia (verified by ECG and elevated TnI and/or CK-MB enzymes), pericarditis, serum Cr>200 µmol/L, coronary artery endarterectomy, left ventricular surgical restoration.

Anesthesia and Operative Technique
Anesthetic induction was performed with midazolam (0.1 mg/kg), hypnomidate (0.2mg/kg), rocuronium bromide (0.6mg/kg and sufentanil (1μg/kg .Maintenance of anesthesia was provided by sufentanil (0.02-0.05 μg/kg/min , sevoflurane (0.8-1.5vol%) and rocuronium bromide (8 -10 μg/kg/min .Standard operative technique through total median sternotomy was used for all patients.Cardiopulmonary bypass (CPB) in condition of mild systemic hypothermia (32 to 34°C) was established with ascending aortic cannulation and right atrial two-stage venous cannulation after systemic heparinization (4 mg/kg) with a target activated clotting time greater than 480 s.Standard management included membrane oxygenators, roller pump with a non-pulsatile flow of 2-2.4 L/min/m 2 with a mean arterial blood pressure around 60 mmHg.After the CPB was discontinued, heparin was neutralized with 0.8 mg protamine sulfate per 1 mg provided.Cell saver and tranexamic acid (30mg/kg) were routinely used.Myocardial protection was achieved by one of the two study solution as antegrade intermittent cold crystalloid cardioplegia (4-8 o C) and topical cooling with iced saline "slush".Cardioplegic solution was administrated as follows: St.Thomas M induction dose was 1000 mL over 3-5 minutes (min) with maintenance doses of 200 mL over 2 min every 20 min thereafter.Custodiol was delivered as one single dose of 20mL/kg over 6-8 min whereas the second dose was provided only when the cross-clamp time exceeded 120 min.If the heart exhibited electrical or mechanical activity during the procedure, additional doses of 200 mL of the cardioplegic solution was administered.The composition of modified St Thomas 5 cardioplegia routinely used in our Institution is: Na + 147mM/L, K + 20mM/L, Mg 2+ 16mM/L, Ca 2+ 2mM/L, Cl - 203mM/L, NHCO3 -10mM/L, Osmolality 388mOsm/kg, pH(25 0 C)~7.8; and the composition of the Custodiol 7 is: Na + 15mM/L, K + 9mM/L, Mg 2+ 4mM/L,Ca 2+ 0.015mM/L, Histidine 198 mM/L, Tryptophan 2mM/L, Ketoglutarate 1mM/L, Mannitol 30mM/L, Osmolality 310 mOsm/kg, pH(25 0 C) ~7.8, pH(25°C) ~7.02-7.20.Perioperative transfusion, fluid administration, and use of inotropes and vazopressor were carried out at the discretion of the anesthesiologists.As primary outcome measures we compared:

Data Collection and Definitions
ECG. Twelve-lead electrocardiogram (ECG) was obtained routinely preoperatively, at Intensive Care Unit (ICU) at arrival and daily until ICU discharge, and whenever the clinical situation of the patient required it.All the ECGs were compared with the preoperative recording for evidence of new postoperative infarction.Heart rhythm and rate were monitored continuously with telemetry during the ICU stay.Patients who suffered at least one episode of atrial fibrillation postoperatively, without history of atrial fibrillation preoperatively, and needed medical treatment were recorded.TTE.Echocardiographic examinations were performed preoperatively and about 24 hours post-surgery by two highly experienced echocardiographers.LVEF was estimated by biplane Simpson's method.Regional wall motion abnormalities were visually assessed and were marked as akinetic, dyskinetic, or hypokinetic segments.Occurrence of new postoperative segmental wall motion defects or deterioration of the existing one, compared with the preoperative echocardiography, were registered.Left ventricle walls thickness more than 11 mm was considered as a marker of left ventricular hypertrophy.Cardiac markers.Cardiac enzymes (cTnI, CKMB) were sampled from peripheral blood preoperatively (T0), at 6 (T6), 24 (T24) and 48 (T4) hours postoperatively according to profile of enzymes release after on-pump CABG 6 .cTnI and CKMB were measured quantitatively by means of enzyme electrochemiluminescence immunoassay (Beckman Coulter Access 2 Analyzer): the upper normal reference limit (URL) (99th percentile) for cTnI is 0.05 µg/L and for CKMB these are 7.2 µg/L male /3.4 µg/L female.Myocardial Infarction Criteria.Peak of cardiac troponin I(cTnI) value > 3.1 µg/L within 48 hours after operation, with normal preoperative values, associated with either: a) Electrocardiogram (ECG) showing new pathological Q waves or new left bundle branch block (LBBB) or b) TTE revealing new regional hypokinetic or akinetic area in the left or right ventricle 7,8 .Acute Kidney Injury (AKI) defined as increase in serum creatinin to ≥ 2 times baseline Statistical analysis.Statistical analysis was performed using the IBM SPSS statistics for Windows, version 20.0 (SPSS Inc., Chicago, IL, USA).Continuous variables were reported as mean and standard deviation or median and interquartile range, whilst categorical variables were given as absolute values and percentages.
A comparison of the two groups, i.e. two clinical treatments, was done using t-test and Mann-Whitney U-test for continuous variables and Chi square test for categorical variables.P values P < 0.0 5 were considered statistically significant.

Results
The study enrolled 104 pts of which 54 pts used Custodiol and 50 pts used St. Thomas M cardioplegia for myocardial protection.Patients in both groups were well balanced with regard to the preoperative demographic and clinical characteristics presented in Table 1.Demographics data were similar, two-thirds of them were male, with a mean patient age about 65 years.There were no statistically significant differences between the subjects in any of the following variables displayed in Table 1: risk factors, non-cardiac and cardiac comorbidities.In terms of ischemic heart diseases, majority of patients had a three vessel coronary artery disease with medium to high degree of coronary disease extensity with regard to average value of SYNTAX score of 29.Furthermore, about half of patients suffered from moderate anginal disorders, class 2, according to CCS (Canadian Cardiovascular Society) Angina Grading Scale.Most of the patients had previously survived one myocardial infarction with preserved LV function and EF greater than 55%.They were categorized to the 2 nd stage of heart failure according to the NYHA (New York Heart Association) classification.Left ventricular hypertrophy was three times more common in the Custodiol group than in the St. Thomas M group (18.5% vs. 6.0%), but it didn't reach statistical significance (p=0.103).We note that hypertrophied myocardium is more vulnerable to ischemic damage 1, 6 .Patients in both groups were consider to be at low surgical risk on the basis of log Euro Score II (European System for Cardiac Operative Risk Evaluation) with mean value of around 1. Considering preoperative therapy, patients in St. Thomas M group notably more often used Preductal compared with patients in Custodiol group, 30.0% vs. 9.3% respectively (p=0.015).Preductal has been proven as cardioprotective agents that reduce ischemia-reperfusion injury of the heart. 9

Baseline characteristics and patient comorbidities
Table 1 Table 2 presents the operative data and postoperative outcomes across the two groups.With regarding to the operative data the average cardiopulmonary bypass time was longer for the Custodiol group compared to the St. Thomas M cardioplegia group, 82.2±23.7 vs. 74.5±18.5 minutes, respectively.This difference trended toward statistical significance with a p-value of 0.075.The cross-clamp time difference between the groups reach statistical significance with a p-value of 0.022 and were considerably longer for patients in the Custodiol group 49.1 ± 19.0 minutes compared to 41.0 ± 12.,9 minutes for the St. Thomas M group.On the other hand, the number of grafts was similar, about 3 grafts per patient, and the internal mammary artery was used in over 95% of cases in both groups.Intraoperative parameters of cardioplegic efficiency, time to cardiac arrest, and spontaneous rhythm recovery rate were similar in both groups.The total amount of used cardiplegia was significantly lager in the Custodiol vs. St.Thomas M group, 1667 vs. 1306 mL, respectively, (p=0.000).Postoperative complications data presented in Table 2, are comparable between the groups.With regard to postoperative clinical indicators of myocardial protection efficacy, the results were as follows: there were neither 30-day deaths nor prolonged MV over 24 hours in either group, the Custodiol group showed less frequently AF (20.4% vs. 28.0%;p=0.496),MI (1.8% vs. 10.0%;p=0.075) and inotropic support (9.0% vs. 12.0%; p=0.651), but without statistical significance, LVEF reduction was comparable (0.81% vs. 1.26%; p=0.891), prolonged ICU and hospital length of stay were less frequent in the Custodiol group, but with no statistical significance, 14.8% vs. 24.0%(p=0.348) and 7.4% vs. 12.0%(p=0.645),respectively.

Operative data and postoperative outcomes
Table 2 In terms of cardiac enzymes, as markers of myocardial necrosis, no statistically significant difference was found in any of the sampling times as shown in Figure 1.The peak of the values of all enzymes was 6 after surgery (see Table 3).

Cardiac enzymes: post-surgery levels at 6 and 24 hours
Table 3 Enzymes values over 48 hours post-surgery (mean ± SD) Figure 1 Composite indicator Parameter of Myocardial Protection Efficacy (PMPE) was introduced to capture occurrence of infrequent clinically important outcomes.In particular, PMPE was defined as occurrence of one of following: postoperative MI, atrial fibrillation de novo, inotropic support, prolonged ICU-LOS>3days, prolonged hospital-LOS>10days, prolonged MV> 24h, 30-Day mortality, cTnI > 8.5 µg/L at 24h post-surgery.Comparison between the groups in terms of PMPE showed a lower rate in the Custodiol group (37,0% vs. 56,0%), which was very close to being statistically significant (p=0.053),(see Table 4).

Discussion
Myocardial injury is an inevitable consequence of cardiac surgery and cardioplegic arrest is the most preferable technique of intraoperative myocardial preservation.There have been numerous studies comparing the effectiveness of myocardial preservation between a wide variety of blood and crystalloid cardioplegia.However, their relative benefits are still a matter of an on-going debate 4 .
Our study compared two strategies of myocardial protection: one using Custodiol and the other using modified St. Thomas cardioplegia.The difference between the studied groups according to the predictors of perioperative myocardial injury 7 and postoperative troponin elevation 6 was not significant except in the case of ACC time and Preductal therapy (see Table 4).In particular, we observed a significantly longer ischemic period, with trends to significantly longer CBP time in the Custodiol group and significantly higher perioperative use of Preductal as a proven cardioprotective agent 9 in the St.Thomas group.Hypertrophied myocardium, which is much more difficult to protect, was considerably more common in the Custodiol group, although without statistical significance.These could have an adverse effect on outcomes in the Custodiol group.

Summarized factors of perioperative myocardial injury
Table 4 Meta-analysis conducted by Edelman et al. 10 compared Custodiol with other conventional crystalloid or blood cardioplegia, with regard to myocardial protection, summarizing fourteen comparative studies, concluding that there was no difference in hospital mortality between Custodiol and other conventional cardioplegia.
Our study shows no mortality, and the reason could be a low preoperative EuroScor II, and infrequent severe myocardial damage, according to relatively low cardiac enzymes levels.Spontaneous rhythm recovery after ACC is commonly used as an indicator of myocardial protection, ranging from 10% to 99%. 11Meta-analysis by Edelman et al. presented statistically significant higher rate of ventricular fibrillation in Custodiol groups (Custodiol 20.1% vs. 9.7%). 10In our study spontaneous rhythm restoration rate was higher in the Custodiol group, 31.5% vs. 20.0% in St Thomas group, but without statistical significance, (p=0.267).No significant difference in the rates of postoperative MI, AF, low cardiac output with inotropic support between groups was found in our study, in agreement with the meta-analysis 10 .We observed five times more frequent MI in the Custodiol group with a trend to statistical significance (p=0.075).In the meta-analysis cross-clamp time was around 60 minutes in both groups and cardiac enzyme levels (CKMB and cTnI) did not differ between groups.The cross-clamp time in our study was under 50 minutes, with comparable enzyme levels between the groups.
There are only few studies in the literature that compared myocardial protection between HTK and St. Thomas cardioplegia [12][13][14][15][16] .These studies discovered no significant difference between the two groups in terms of mortality, however, their findings on myocardial protection in terms of other specific indicators (listed in Table 4) varied in a fashion which seems to suggest that benefits of HTK cardioplegia become more pronounced with longer ACC.In the study by Arslan et al. 12 , which involved shorter clamping time (less than 40 min) the only significant difference found was longer time to cardiac arrest in the HTK group, 63 vs. 54 seconds in the St. Thomas group, that could be a disadvantage because it causes more ischemic-reperfusion damage 10,12 .On the other hand, time to cardiac arrest in our study was of about 60 seconds for both groups, whereas ACC time was about 40-50 min.Demmy et al. 13 demonstrated lower defibrillation rate after ACC (64% vs. 91%), but a significantly higher peak level of cTnI 6 hours after surgery in the HTK group (no information about ACC time was given) .Hamed et al. 14 , in a study with ACC time mean value of about 60 minutes showed that St. Thomas cardioplegia was comparable to HTK and blood cardioplegia in pediatric cardiac surgery.Carega et al. 15 demonstrated improved myocardial protection in adult cardiac surgery with HTK cardioplegia according to all considered indicators at myocardial ischemic time longer than 60 minutes.Lin et al. 16 demonstrated superiority of HTK cardioplegia in complex pediatric cardiac surgery with ischaemic time over 150 minutes 16 .In our study cross-clamp time was below 50 min in both groups, although significantly longer in the Custodiol group, and myocardial protection was comparable between the groups.
Troponin and CKMB are routinely used to evaluate the degree of myocardial damage associated with cardiac surgery.Cardiac troponin I has been shown to be the most sensitive biochemical marker of intraoperative myocardial injury and is therefore an valuable indicator of the quality of myocardial protection 6,17 .It has shown that in uncomplicated cardiac surgery, there is an early increase of cardiac troponin I around 6 hours post surgery, followed by a rapid decrease, falling down to substantially lower concentrations at 24 hours.A later release of cTnI is more indicative of severe myocardial damage.The unfavorable outcome is indicated if cTnI peaked > 8.5 µg/L 24 hours post surgery 17-19 .Postoperative release of cardiac enzymes in our study (see Table 3) over 48 hours, reached peaked values of around 5µg/L at 6 hours post surgery in each group, indicating a lesser perioperative myocardial damage.This result is in accordance with previous studies 12- 14 .PMPE variable revealed a higher rate of adverse outcomes of poor myocardial protection in the St.Thomas M group, very close to statistical significance (p=0.053).

Conclusion
Custodiol is safe and as effective as conventional cold crystalloid modified St.Thomas cardioplegia for myocardial protection in CABG surgery.The considerably less frequent MI, with a trend towards statistical significance, despite the significantly longer cross-clamp time in the Custodiol group may suggest that its benefits even in operations with shorter ischemic time could be ascertained in a larger study.


Troponin I (cTnI) levels preoperative, 6, 24 and 48 hours post surgery  Changes in Left Ventricul Ejection Fraction (LVEF) by Transthoracic Echocardiogram (TTE), 24 hours post surgery As a secondary outcome measures, we compared:  30-day mortality  Creatine Kinase Isoenzyme MB (CKMB) levels preoperative, 6, 24 and 48 hours post surgery  Time to cardiac arrest: time elapsed from the introduction of cardioplegic infusion to the cessation of cardiac electrical and mechanical activity  Spontaneous rhythm restoration post aortic cross-clamp (ACC)  Prolonged echanically Assisted Ventilation ≥ 24h, up to 48 hours post surgery  Inotropic Support ≥ 60 min, up to 48 hours post surgery  Myocardial Infarction, up to 48 hours post surgery  Atrial fibrillation de novo, up to 48 hours post surgery  Prolonged Intesive Care Unit (ICU) and hospital Length of Stay (LOS) > 3 days  Other postoperative morbidity (infection-deep wound infection or sepsis, stroke, acute kidney injury, length of hospitalization)