Vasculoprotective role of del Nido cardioplegia in open-heart surgery via modulation of E-Selectin, Endocan, and TNF-alpha coronary sinus blood samples

Abstract

Purpose

Cardioplegia is routinely used in cardiac surgery to prevent endothelial damage associated with ischemia-reperfusion. However, there is no consensus on the optimal vasculoprotective cardioplegia. We aim to assess endothelial integrity using coronary sinus blood samples in patients who have received various types of cardioplegia.

Methods

Aortic valve replacement for aortic stenosis and isolated coronary artery bypass grafting patients were included. del Nido and blood cardioplegias were subjected to assessment. Blood samples from the coronary sinus were collected before and after aortic cross-clamping. Endothelial and redox biomarkers were assessed utilizing immunochemical and colorimetric techniques.

Results

In coronary artery bypass grafting patients, E-selectin levels were found significantly lower after cross-clamping compared to pre-clamp values, regardless of cardioplegia type (p = 0.015). ROC analysis revealed that Endocan, E-selectin, and TNF-α levels are reliable biomarkers of endothelial injury after cardiopulmonary bypass (p values: 0.004, 0.007, and < 0.001 for each parameter, respectively). Total oxidant status emerged as a significant redox biomarker, with 87% ROC sensitivity (p = 0.037).

Conclusion

This study uniquely assesses localized endothelial responses via coronary sinus sampling with a multiplex biomarker panel, providing evidence of del Nido cardioplegia’s protection. Our findings indicate that del Nido cardioplegia exhibits significant vasculoprotection, as evidenced by modulation of coronary sinus Endocan, E-selectin, and TNF-α. These biomarker changes, together with the absence of adverse differences in major postoperative outcomes, suggest that del Nido cardioplegia provides efficient myocardial and vascular protection in both aortic valve replacement and coronary artery bypass procedures, supporting its safe use in adult elective cardiac surgery.

Introduction

Just as the heartbeat is considered the most fundamental sign of life, both physiological and pathological states of the heart can lead to systemic consequences throughout the body. Therefore, successful outcomes in cardiac surgery rely not only on the efficacy of the surgical procedure but also on mitigating its possible harmful systemic consequences. Cardiopulmonary bypass (CPB), a key part of cardiac surgery, is known to cause various systemic and localized inflammatory responses [1]. Alongside inflammation, perioperative care must consider pathophysiological processes such as endothelial dysfunction, leukocyte adhesion, and increased vascular permeability.

Myocardial protection is a prominent focus of research in modern cardiac surgery. The recognition that CPB alone is inadequate for maintaining a steady and bloodless operational field prompted the innovation of cardioplegia, developed by Melrose and colleagues [2]. Since that time, numerous myocardial protection strategies have been implemented to maintain the heart in an arrested state for the maximum duration while minimizing myocardial damage. Among these strategies, del Nido cardioplegia was initially used in pediatric cardiac surgery but has gained increasing popularity in adult cardiac procedures as well [3]. The number of studies on del Nido cardioplegia has risen alongside its broader application. Its ability to provide extended cross-clamp durations, facilitate continuous surgical operations, and demonstrate non-inferiority compared to traditional blood cardioplegia in several investigations has led to its growing endorsement by cardiac surgeons [4, 5].

The non-physiological environment of CPB and the inevitable diastolic arrest cause increased systemic production of reactive oxygen species (ROS) and oxidative damage, leading to extensive inflammatory responses that need to be clinically considered [6]. Redox dyshomeostasis, primarily caused by ROS overproduced by inflammatory cells in the vasculature and myocardium, disrupts the redox homeostasis of plasma proteins and blood cells after cardiac surgery [7]. This redox imbalance directly leads to postoperative organ dysfunction, rendering the tracking of oxidative load and the application of redox modulatory strategies essential.

Contact between blood and non-endothelial surfaces during CPB is a primary cause of ROS overproduction. Factors include non-pulsatile flow, cross-clamping, anesthetic agents, myocardial injury, complement activation, and reperfusion, all of which induce systemic oxidative damage [8]. Furthermore, neutrophils, catecholamines, cytokines secreted by active neutrophils, endothelial damage, the kallikrein cascade, and endotoxin release play a role in ROS-mediated inflammatory responses. The resultant endothelial injury and consequent vascular instability may result in postoperative organ and systemic dysfunction, highlighting the necessity for adequate precautions.

Atherosclerosis, the fundamental pathophysiological process underlying coronary artery disease (CAD), exacerbates vascular dysfunction following surgery [9]. Aortic stenosis is similarly linked to atherosclerotic alterations that affect the arterial bed, diminish vascular compliance, and lead to endothelial damage [10]. Moreover, the hypertrophied myocardium resulting from aortic stenosis is more vulnerable to ischemia, thereby amplifying the necessity for myocardial protection in this group of patients. Therefore, examining the effects of vascular pathophysiology and the effectiveness of myocardial protection is of considerable therapeutic importance.

Although previous studies have assessed the oxidative effects of CPB and myocardial protection using cardiac and redox biomarkers, few have evaluated the influence of different cardioplegias on ROS-mediated endothelial damage and vasculoprotection [11]. Endothelial cells are the primary source of endocan, also referred to as endothelial cell-specific molecule-1, a soluble proteoglycan. Tumor necrosis factor-alpha (TNF-α) and other ROS-mediated inflammatory stimuli can increase endocan expression [12]. In vascular diseases, E-selectin mainly regulates the physiological adherence of leukocytes to the endothelium [13]. E-selectin is also constantly expressed in response to ROS-mediated inflammation, although it remains essentially inactive under physiological conditions [14]. This study aims to assess the vasculoprotective effect of del Nido cardioplegia. Consequently, we recruited patients with CAD, characterized by prevalent endothelial dysfunction and atherosclerosis, alongside patients with aortic stenosis, whose hypertrophied myocardium exhibits increased susceptibility to ischemic injury. Since studies conducted on coronary sinus blood are known to yield more specific and reliable results, we evaluated alterations in the levels of the endothelial biomarkers such as E-selectin, endocan, and TNF-α, as well as redox biomarkers, by analyzing coronary sinus samples to assess the vasculoprotective characteristics of del Nido and blood cardioplegias [15].

Materials and methods

Patient groups

A prospective study was conducted at Dr. Siyami Ersek Hospital from November 2022 to July 2023. We evaluated patients planned for coronary artery bypass grafting (CABG) and aortic valve replacement (AVR) surgery. Sample size was calculated using G*Power 3.1.9 [16] based on the effect size (Cohen’s d = 0.455, f ≈ 0.23) reported by Federico et al. [17] for endocan levels in four groups. With α = 0.05 and 95% power, the required sample size was n = 88 (22 per group), and to allow for potential dropouts, 23 patients per group were planned. The study was completed with 74 patients (n = 23, 21, 15, and 15, respectively), and a post hoc power analysis performed with G*Power (version 3.1.9) showed an attained effect size of f = 0.74 (large effect according to Cohen’s convention), confirming statistical power exceeding 0.95.

Patients were then categorized into types of cardioplegia groups as del Nido and blood cardioplegias, which were used in the surgeries. Using inclusion and exclusion criteria helped us to create study groups that recruited homogeneous individuals into random groups. To minimize measurement bias, assays were conducted by a single blinded laboratory technician using the immunochemical method for the studied biomarkers. Both participants and clinical staff were blinded to treatment allocation. During data cleaning and analysis, cardioplegia groups were labeled as ‘del Nido’ and ‘Blood.’ The trial statistician, also blinded to treatment codes, accessed them only after the statistical analysis plan was finalized and the database was locked. Unblinding took place after all analyses were completed according to the pre-specified statistical analysis plan. The flow diagram demonstrating inclusion/exclusion criteria for the cardioplegia groups is given in Fig. 1.

Fig. 1.

The flow diagram demonstrating inclusion/exclusion criteria for the final cardioplegia groups

Ethical considerations

The study protocol was approved by the institutional ethics committee (File number: HNEAH-KAEK 2022/43–3507). Informed consent from all the patients for the operations and the study was obtained before the study.

Surgical procedure

Standard surgical protocols for CABG and AVR operations were applied under moderate hypothermia (30 °C). Myocardial protection with moderate hypothermia was also supported with the application of topical cooling using ice slush. The initial dose of blood cardioplegia solution, prepared with blood taken from the cardiopulmonary bypass circuit after the patient was cooled to 30 °C (4 parts blood to 1 part crystalloid), was administered as 1000 mL via the antegrade route, followed by intermittent doses of 400 mL every 20 min, delivered both antegrade and through the saphenous veins [18].

The del Nido cardioplegia solution was prepared by incorporating the following constituents into 1000 cc of Plasmalyte-A solution and stored at + 4 °C: 26 mEq of potassium chloride, 16 ml of 20% mannitol, 4 ml of 50% magnesium sulfate, 13 ml of 8.4% sodium bicarbonate, and 6.5 ml of 2% lidocaine [19] (Table 1).

Table 1.

Comparative characteristics of Del Nido and blood cardioplegias

Feature	del Nido Cardioplegia	Blood Cardioplegia
Main solution	Isolen-S 1000 cc + additional agents	1000 cc blood (from the cardiopulmonary bypass circuit)
Additives	− 20% Mannitol: 16.3 cc - Magnesium Sulfate (8 mEq): 13.4 cc - Sodium Bicarbonate: 13 cc − 2% Lidocaine: 6.5 cc − 22.5% Potassium Chloride: 8.6 cc	− 22.5% Potassium Chloride: 8.5 cc
Preparation method	200 cc of blood taken from the cardiopulmonary bypass circuit mixed with prepared solution (1:4)	800 cc prepared blood cardioplegia mixed with 200 cc Plasma-lyte-A (4:1)
Administered volume (Initial dose)	1000 cc solution	1000 cc solution
Temperature	+ 4 °C	+ 30 °C
Route of administration	Antegrade (aortic root)	Antegrade (aortic root)
Re-dosing frequency	60 min	20 min
Administered volume (Maintenance dose)	500 cc (including 100 cc added blood)	400 cc (with an equal proportion of potassium)

The solution was administered as the standard antegrade route in both AVR and CABG patients with an initial 1000 mL followed by a 500 mL maintenance dose every 60 min. In clinical practice, most surgeons will re-dose del Nido cardioplegia between 60 and 90 min of ischemic time, with a dose ranging from 250 to 500 mL chosen based on the anticipated remaining length of cross-clamp time [20, 21]. In patients undergoing CABG utilizing del Nido cardioplegia, anastomotic control was achieved with 5–10 cc of del Nido solution. Following the completion of distal coronary anastomoses, proximal anastomoses were executed with aortic side clamping.

Transesophageal echocardiography was used to determine perioperative gradients, assess valve incompetence, and evaluate the presence of paravalvular leaks. Graft flows were evaluated by Doppler ultrasonography perioperatively.

Coronary sinus blood sampling

CPB commenced with standard procedures, utilizing aortic and venous cannulation. Before CPB started, while the heart was still beating, the retrograde catheter was precisely inserted into the right atrium through a small incision secured with a purse-string suture. Meanwhile, the surgeon retracted the heart cephalad to expose the atrioventricular groove. Using the right hand, the gently curved retrograde catheter was gently engaged in the coronary sinus. The catheters’ placement was confirmed through transesophageal echocardiography. After securing the catheter with an inflated balloon and expelling air, a 2 cc sample was stored for analysis. Coronary sinus blood samples were collected before cross-clamping and 4 min after cross-clamp removal [22].

Blood sample processing and storage

Blood samples taken from the coronary sinus were processed into serum by centrifugation within 45 min of collection. This minimizes the risk of hemolysis, platelet activation, and changes in biomarker levels [23]. Serum samples were obtained by centrifuging blood collection tubes containing coronary blood samples without additives for 15 min at 3000 rpm and 4 °C. Serum aliquots were frozen and stored at − 80 °C until they were analyzed for tissue integrity and redox biomarkers.

Vascular biomarker assays

We assessed the effects of different cardioplegias on cardiac protection by analyzing coronary sinus tissue integrity and redox biomarkers using immunochemical and spectrophotometric methods during pre- and post-operative periods (Table 2).

Table 2.

Assayed endothelial integrity and redox biomarkers

Biomarker groups	Assayed biomarkers	Features of assayed biomarkers	Analytic method
Endothelial biomarkers	E-Selectin	Upregulated expression in response to stimulation by pro-inflammatory cytokines. ROS overproduction contributes to endothelial cell damage and abnormal E-selectin expression [13].	ELISA (Shanghai Korain Biotech Co, Shanghai, China)
	Endocan	Higher Endocan is an independent predictor of the presence of diminished coronary flow. Patients with atherosclerotic stroke exhibit notably higher serum endocan levels [24]	ELISA (Shanghai Korain Biotech Co, Shanghai, China)
	TNF-alpha	The expression of TNF-α is stimulated by atherogenesis, myocardial ischemia, and impaired redox status [25, 26]	ELISA (Shanghai Korain Biotech Co, Shanghai, China)
Redox biomarkers	Total oxidative status	Its higher levels indicate oxidative damage, inflammation, and various diseases such as atherosclerosis, cancer, and diabetes [27, 28]	Colorimetric
	Total antioxidative status	Higher levels reflect the ROS buffering effect of all antioxidants present in plasma and body fluids [27]	Colorimetric
	Oxidative Stress Index	Oxidative Stress Index, which is considered an indicator of oxidative stress, is expressed as the percentage ratio of Total Oxidant Status to Total Antioxidant Status [29]	Calculated index

Routine clinical chemistry assays

All patients had preoperative clinical chemistry test results, including a whole blood count and a wide range of routine parameters. Additionally, all these parameters were also assayed after surgery, as well as on the 2nd and 5th days.

Demographic data

The demographic data of the patients in each cardioplegia group are given in Table 3.

Table 3.

Demographic data, comorbidities, and echocardiographic findings

	del Nido cardioplegia (n = 38)	Blood cardioplegia (n = 36)	p
Gender			0.88
Male	27.0 (71.1%)	25.0 (69.4%)
Female	11.0 (28.9%)	11.0 (30.6%)
Age	62.0 (57.2–68.8)	64.0 (57.0–70.0)	0.73
BMI	27.4 ± 5.3	27.7 ± 3.7	0.74
DM			0.89
Insulin	7.0 (18.4%)	6.0 (16.7%)
OAD	12.0 (31.6%)	10.0 (27.8%)
None	19.0 (50.0%)	20.0 (55.6%)
Hyperlipidemia	20.0 (52.6%)	17.0 (47.2%)	0.64
Hypertension	31.0 (81.6%)	28.0 (77.8%)	0.68
CKD	8.0 (21.1%)	6.0 (16.7%)	0.63
CAD	7.0 (18.4%)	6.0 (16.7%)	0.84
COPD	4.0 (10.5%)	8.0 (22.2%)	0.17
Smoking	24.0 (63.2%)	21.0 (58.3%)	0.67
Euroscore II	0.95 (0.76–1.32)	1.00 (0.87–1.55)	0.42
SYNTAX 2	25.0 (22.6–32.2)	24.7 (19.8–31.4)	0.57
EF	60.0 (46.2–60.0)	55.0 (50.0–60.0)	0.60
LVEDD	47.0 (44.0–51.0)	47.5 (46.0–53.2)	0.64
LVESD	29.0 (26.0–35.5)	31.0 (27.8–34.2)	0.25
LVMI	113.5 (88.5–134.2)	108.5 (94.8–128.2)	0.84

*BMI: Body mass index, DM: Diabetes mellitus, OAD: Oral antidiabetic, CKD: Chronic kidney disease, CAD: Carotid artery disease, COPD: Chronic obstructive pulmonary disease, EF: Ejection fraction, LVEDD: Left ventricular end-diastolic diameter, LVESD: Left ventricular end-systolic diameter, LVMI: Left ventricular mass index

There was no significant difference among the demographic data of patients and their respective controls concerning age, gender, comorbidity, and echocardiographic data. In the CABG cohort, SYNTAX scores were calculated from the coronary angiography images [30]. The insignificant data on demographics and comorbidities given in Table 2 indicate that our cardioplegia-related experimental findings are independent of the patients’ demographics.

Statistical analysis

G-Power (version 3.1.9) was used to determine the sample size for the study groups. To conduct the statistical analysis of the data, SPSS (version 29.0) was employed. The mean ± standard deviation (mean ± SD) was provided to express the descriptive statistics in our study. The normality of the data distribution was assessed using the Shapiro-Wilk test. Non-normally distributed data for parametric variables were analyzed using the non-parametric Mann-Whitney U test and the related-samples Wilcoxon signed-rank test, while normally distributed data were examined using the parametric independent-samples t-test and paired samples test. To account for baseline differences in biomarkers, ANCOVA was performed using pre-clamp values as covariates. Post hoc pairwise comparisons were corrected for multiple testing using the Holm–Bonferroni method. Correlation analyses were conducted using Pearson’s correlation coefficient for normally distributed data and Spearman’s correlation coefficient for non-normally distributed data. P values of < 0.05 were regarded as indicating statistical significance, with a confidence level of 95%. The sensitivity and specificity of the assayed biomarkers were assessed using the receiver operating characteristic (ROC) curve analysis.

Results

Operative findings

The information gathered during the surgery included how long the CPB lasted, how long the aortic cross-clamp (CC) was in place, the need for inotropic support, how often ventricular fibrillation occurred, and the need for mechanical support, which are shown in Table 4. The clinical findings did not exhibit any significant differences.

Table 4.

Operative findings

	del Nido cardioplegia (n = 38)	Blood cardioplegia (n = 36)	p
CPB time	117.1 ± 28.6	112.7 ± 36.3	0.56
CC time CC – CPB time	78.3 ± 24.2 19.5 (15.0–30.0)	78.4 ± 27.6 25.0 (18.5–34.5)	0.98 0.14
Inotropes			0.85
0	6.0 (15.8%)	7.0 (19.4%)
1	23.0 (60.5%)	20.0 (55.6%)
2	8.0 (21.1%)	6.0 (16.7%)
3	1.0 (2.6%)	2.0 (5.6%)
4	0.0 (0.0%)	1.0 (2.8%)
VF Spontaneous rhytm	3.0 (7.9%) 32.0 (88.9%)	4.0 (11.1%) 35.0 (92.1%)	0.70 0.43
IABP need	1.0 (2.6%)	1.0 (2.8%)	> 0.99
CPB need	2.0 (5.3%)	3.0 (8.3%)	0.67

*CPB: Cardiopulmonary Bypass, CC: Aortic Cross-Clamp Time, CC-CPB: Time from Cross-Clamp Removal to Bypass Termination, VF: Ventricular Fibrillation, IABP: Intra-aortic Balloon Pump

Vascular and redox biomarker-related findings

In CABG patient groups treated with both types of cardioplegia, post-cross clamp Endocan, E-selectin, TNF-α, and total oxidant status (TOS) levels were lower compared to pre-cross clamp values, but there was a significant decrease in, TNF-α, total oxidant status (TOS) levels, total antioxidant status (TAS), and oxidative stress index (OSI) was observed in post-cross-clamp coronary sinus blood samples from the CABG surgery group who received del Nido cardioplegia (Table 5).

Table 5.

Intragroup comparisons for vascular and redox biomarker-related findings in the coronary sinus blood for CABG patients

Parameters	CABG Group (n = 44)
	del Nido cardioplegia (Pre-clamp) (n = 23)	del Nido cardioplegia (Post-clamp) (n = 23)	Blood cardioplegia (Pre-clamp) (n = 21)	Blood cardioplegia (Post-clamp) (n = 21)
E-Selectin (ng mL⁻¹)	0.025 ± 0.005	0.022 ± 0.005*	0.028 ± 0.007	0.026 ± 0.005
Endocan (ng mL⁻¹)	0.842 ± 0.318	0.836 ± 0.345	0.704 ± 0.232	0.586 ± 0.156*
TNF-α (ng mL⁻¹)	0.187 ± 0.020	0.125 ± 0.009*	0.175 ± 0.023	0.143 ± 0.016*
TOS (µmol H₂O₂ eq L⁻¹)	13.14 ± 1.90	10.25 ± 1.88*	13.39 ± 2.18	11.33 ± 1.85*
TAS (mmol L⁻¹)	0.83 ± 0.13	1.02 ± 0.11*	0.93 ± 0.24	1.01 ± 0.13*
OSI (AU)	16.33 ± 4.46	10.14 ± 2.28*	15.68 ± 5.86	11.47 ± 2.92*

*=pre- vs. post-CPB sinus blood and p < 0.05 values are shown in bold. TNF- α: Tumor necrosis factor-alpha, TOS: Total oxidant status, TAS: Total antioxidant status, OSI: Oxidative stress index

No significant difference was noted for E-selectin and TAS in the blood cardioplegia group. Endocan levels in CABG patients with del Nido were significantly lower in the blood cardioplegia group, coronary sinus blood samples, than in patients with del Nido.

In the AVR group, post-cross-clamp coronary sinus blood samples showed a significant decrease in endocan levels with both cardioplegia solutions. No significant differences were observed for E-Selectin, TNF-α, TOS, and TAS between the two cardioplegia groups. However, OSI levels were significantly lower in the del Nido cardioplegia group compared with blood cardioplegia, indicating a relative reduction in oxidative stress with del Nido solution (Table 6).

Table 6.

Intragroup comparisons for vascular and redox biomarker-related findings in the coronary sinus blood for AVR patients

Parameters	AVR Group (n = 30)
	del Nido cardioplegia (Pre- clamp) (n = 15)	del Nido cardioplegia (Post- clamp) (n = 15)	Blood cardioplegia (Pre- clamp) (n = 15)	Blood cardioplegia (Post- clamp) (n = 15)
E-Selectin (ng mL⁻¹)	0.034 ± 0.003	0.030 ± 0.004*	0.035 ± 0.003	0.032 ± 0.004*
Endocan (ng mL⁻¹)	0.671 ± 0.210	0.594 ± 0.167*	0.586 ± 0.113	0.513 ± 0.092*
TNF-α (ng mL⁻¹)	0.239 ± 0.027	0.224 ± 0.010	0.230 ± 0.026	0.219 ± 0.013
TOS (µmol H₂O₂ eq L⁻¹)	12.16 ± 2.23	9.49 ± 1.66*	12.04 ± 2.35	9.89 ± 1.90*
TAS (mmol L⁻¹)	0.83 ± 0.15	0.90 ± 0.12	0.91 ± 0.10	0.92 ± 0.18
OSI (AU)	15.18 ± 3.99	10.66 ± 1.91*	13.29 ± 2.74	11.21 ± 3.25

*=pre- vs. post-CPB sinus blood and p < 0.05 values are shown in bold. TNF- α: Tumor necrosis factor-alpha, TOS: Total oxidant status, TAS: Total antioxidant status, OSI: Oxidative stress index

To account for potential baseline differences in biomarker levels, ANCOVA analyses were performed with pre-clamp values as covariates in the CABG group. For TOS, both the covariate (F(1,41) = 31.56, p < 0.001) and cardioplegia type (F(1,41) = 4.76, p = 0.035) were significant. Adjusted means indicated higher TOS in the blood cardioplegia group (11.26 ± 0.31) compared to del Nido (10.32 ± 0.30). For E-Selectin, baseline values were significant (p < 0.001), but the group effect did not reach significance (F(1,41) = 3.56, p = 0.066). Endocan levels were significantly higher in del Nido compared to blood cardioplegia after adjustment (F(1,41) = 7.34, p = 0.010). TNF-α showed the strongest group difference (F(1,41) = 38.56, p < 0.001), with adjusted means of 144.7 vs. 123.0 (Table 7). In the AVR group, no significant differences were observed for any biomarker.

Table 7.

Post-clamp vascular and redox biomarker levels by cardioplegia type in CABG group: ANCOVA-adjusted means, mean differences, and statistical significance

Biomarkers (post-clamp)	Covariate Effect (F, p)	Group Effect (F, p)	Adjusted Mean Blood Cardioplegia (SE, 95% CI)	Adjusted Mean Del Nido Cardioplegia (SE, 95% CI)	Mean Difference (Blood cardioplegia-Del Nido cardioplegia)	Significance
E-Selectin (ng mL⁻¹)	F(1,41) = 20.95, p < 0.001	F(1,41) = 3.56, p = 0.066	25.62 (0.98, 23.65–27.59)	23.03 (0.93, 21.15–24.92)	2.58	ns
Endocan (ng mL⁻¹)	F(1,41) = 0.94, p = 0.339	F(1,41) = 7.34, p = 0.010	596.8 (60.4, 474.8–718.8)	826.4 (57.6, 710.0–942.8)	–229.6	p < 0.05
TNF-α (ng mL⁻¹)	F(1,41) = 14.73, p < 0.001	F(1,41) = 38.56, p < 0.001	144.7 (2.49, 139.7–149.7)	123.0 (2.38, 118.2–127.8)	21.7	P < 0.001
TOS (µmol H₂O₂ eq L⁻¹)	F(1,41) = 31.56, p < 0.001	F(1,41) = 4.76, p = 0.035	11.26 (0.31, 10.63–11.88)	10.32 (0.30, 9.72–10.92)	0.94	P < 0.05

Post-aortic unclamping biomarker levels were adjusted for baseline (pre-clamp) values using ANCOVA. Adjusted means (± SE) with 95% confidence intervals are presented for Blood and Del Nido cardioplegia groups. Mean differences between groups (Blood – Del Nido) and statistical significance (p-values) are reported. Positive mean difference indicates higher values in the Blood cardioplegia group. Covariate effects of baseline values are reported in the ‘Covariate Effect’ column. p < 0.05 = significant; p < 0.001 = highly significant; ns = not significant

SE = standard error; CI = confidence interval; ns = not significant; TOS = total oxidative status; TNF-α = tumor necrosis factor-alpha

Evaluation of the risk sensitivity of the studied biomarkers

ROC plot graphics involve plotting pairs of sensitivity values of CPB patients with blood cardioplegia versus (1 − specificity) values of CPB patients with del Nido at all possible values for the decision threshold when sensitivity and specificity are calculated nonparametrically. The ROC analysis graph for vasculoprotective endothelial biomarkers and oxidation status is given in Fig. 2.

Fig. 2.

ROC analysis graph for vasculoprotective endothelial biomarkers and oxidation status parameter

ROC curve analyses were conducted to evaluate biomarker performances in both patient groups for del Nido cardioplegia. The Area Under Curve (AUC) value is a comprehensive statistic of the ROC curve that indicates the test’s capacity to differentiate between patient groups. AUC values span from 0.5 to 1.0, where a value of 0.5 signifies that the test performs no better than random chance in differentiating between the groups. A value of 1.0 signifies flawless discrimination. AUC values above 0.80 are typically regarded as clinically valuable, whilst values below 0.80 are deemed to possess little clinical relevance. The diagonal reference line at a 45° angle indicates the diagnostic test’s discriminative ability, comparable to random chance. The upper left corner signifies optimal discriminatory power, characterized by a true positive fraction of 1 and a false positive fraction of 0, indicating both sensitivity and specificity reach 100% [31]. According to the ROC analysis, the endothelial biomarkers studied were found to be diagnostically sensitive risk biomarkers in post-cross-clamp blood samples in coronary artery bypass graft surgery patients with del Nido. This finding emphasizes the diagnostic risk assessment utility of TOS in assessing the detrimental effects of CPB on redox status in blood samples, which exhibit similar trends.

Correlations

The correlation analysis between the endothelial and oxidation biomarkers in the blood samples from the coronary sinus taken before and after the cross-clamp with both cardioplegias was evaluated. In the blood cardioplegia group, there was a positive correlation between TOS and OSI (r = 0.89, p < 0.001) in the CABG cohort and a negative correlation between TOS and E-selectin (r = −0,62, p:0.014) in the AVR cohort. A negative correlation was found between total antioxidant status (TAS) and oxidative stress index (OSI) within the CABG group (r = −0,78, p < 0.001). In the del Nido group, there was also a positive correlation between TOS and OSI (r = 0,83, p < 0.001). A negative correlation was also found between TAS and OSI within the same group (r = −0,48, p:0,022).

Routine clinical chemistry parameters

The routine clinical chemistry parameters in venous blood samples obtained post-CPB in forty-four patients undergoing coronary artery bypass surgery and thirty patients undergoing aortic valve replacement were assayed. No significant difference was found between patient groups. Data was not shown.

Discussion

Cardioplegias are routinely used in cardiac surgery to protect the heart from CPB-mediated endothelial injury. del Nido cardioplegia offers myocardial protection for extended periods, thus reducing the duration of aortic cross-clamping, CPB, and the volume of cardioplegia solution needed [22]. It is also debatable whether del Nido is superior to other cardioplegias in terms of myocardial protection when considering systemic and cardiac biomarkers; some promising vasculoprotective effects have been reported previously [32, 33]. It is widely believed that conventionally used drugs and pharmacological agents can cause unwanted post-surgical effects in cardiovascular practice, making drug interactions a common subject of investigation [34]. Supporting this, the lack of significance in our operative parameters suggests that the observed variations in biomarker levels are not due to operative outcomes. Instead, these clinical findings may result from differences in the composition of the cardioplegic solutions used.

Examining the effects of vascular pathophysiology and the effectiveness of myocardial protection with cardioplegias is of considerable clinical importance. Dysregulation of the systemic levels of E-selectin has been commonly seen in several cardiovascular pathologies [35–38]. ROS-mediated oxidation of plasma constituents induces endothelial cell damage, leading to aberrant expression of E-selectin. E-selectin interacts with specific carbohydrate ligands on the surface of leukocytes to promote their adherence to the endothelium during inflammation. Furthermore, the correlation between higher levels of E-selectin and the increased magnitude of inflammation needs to be accounted for in cardioplegia selection. The selection of cardioplegia solution is important for interrupting the ROS-induced inflammatory cascade at its beginning, and this E-selectin/E-selectin ligand axis is a prime target [13].

ROC analysis is a valuable technique for illustrating the relationship between the sensitivity and specificity of systemic redox biomarkers [11]. The significant decrease in coronary sinus E-selectin levels and its ROC assessment results with high sensitivity and specificity in post-cross-clamp coronary sinus blood samples of the coronary artery bypass graft surgery patient group with del Nido cardioplegia further support its clinical potential as a risk biomarker. Our E-selectin-related findings are providing a non-invasive approach for assessing endothelial protection status. However, the specificity and sensitivity of E-selectin as a diagnostic or prognostic risk indicator may vary across different cut-off values.

Numerous cardiovascular and metabolic diseases, such as Type 2 diabetes, hypertension, atherosclerosis, and coronary artery disease, are closely linked to elevated endocan levels [39]. On the other hand, studies are reporting that different group of drugs causes a changes in serum endocan levels, such as statins, antihypertensive drugs [12, 40] and antidiabetics like metformin [41, 42]. Considering the complexity of the endocan-related vascular processes, we believe that differences in serum endocan levels cannot be solely linked to variations of the cohorts. Moreover, we thought that high serum endocan levels in our specific cohort groups may be linked to the limited effectiveness of the pharmacological treatment regimens and the increasing impact of some other drugs, like metformin.

Endocan exhibits similar trends to E-selectin with different cut-off values in post-cross-clamp coronary sinus blood samples of the coronary artery bypass graft surgery patient group with del Nido cardioplegia. Assessing other endothelial biomarkers in this group of patients with del Nido, such as TNF-α and Endocan, operating with different cut-off values, is necessary to establish standardized risk assessment thresholds and understand their biomarker value in cardiovascular protection. Elevated circulating endocan is usually related to endothelial damage, inflammation, angiogenesis, and cardiovascular stress. There is currently no direct evidence linking del Nido cardioplegia to increased endocan levels. In an observational study of patients undergoing CABG, serum endocan rose postoperatively, associated with duration of norepinephrine support compared with baseline [43]. The ROC graph reinforced the value of total oxidant status as an endothelial injury-inducing biomarker, showing a similar trend to post-CPB endothelial biomarkers in the coronary artery bypass graft surgery patient group. When evaluating isolated aortic valve surgery patients who underwent del Nido cardioplegia, post-op OSI and Endocan levels were found to be significantly lower compared to pre-op levels. A negative correlation was also found in the AVR patients between post-CBP TOS and E-selectin levels. This negative correlation might indicate a possible reciprocal interaction mechanism for post-CBP TOS and E-Selectin for the emerging detrimental effects of TOS-mediated endothelial injury. In patients with valvular heart disease who underwent blood cardioplegia, the lack of a significant decrease in the OSI index in postoperative blood samples is an indication in favor of del Nido cardioplegia. Additionally, TOS and OSI exhibited a positive correlation in the blood cardioplegia group. In AVR patients with del Nido, the apparent fact is that only TNF-α stands out as an independent clinical risk factor among all biomarkers analyzed with ROC. We believe that high oxidant load may lead to TNF-alpha emerging as a risk factor in ROC. We suggest that TNF-α assessment could be used as an important inflammatory marker in the follow-up of this group of patients with del Nido.

ROS also regulate vascular function by modulating various redox-sensitive signaling mediators. Endocan interacts with these mediators and thereby regulates cell migration, proliferation, and its function during health and disease conditions [44]. A negative correlation in vascular redox biology means that as oxidative stress biomarkers increase, endothelial biomarkers decrease (or vice versa). Compensatory endothelial response, consumption or degradation of endothelial markers, feedback inhibition, and the stage of disease and stress response can be considered as mechanistic explanations [45]. Our study demonstrated that a negative correlation was found between TAS and OSI within the CABG group, which might be related to consumption and/or degradation of antioxidant system components. Studying the pathophysiological features of activated endothelium in cardiovascular and other disorders is made possible by the fact that E-selectin is only expressed on this cell. Nonetheless, plasma contains a soluble form of E-selectin [46]. We also found that there was a negative correlation between TOS and E-selectin in the CABG cohort. We attribute these findings to the impairment in endothelin synthesis in ROS-activated endothelial cells in the CABG group.

Furthermore, in CABG patients, the primary pathophysiological background is coronary artery disease with pre-existing endothelial dysfunction and increased leukocyte-endothelium interaction. E-selectin, as an early indicator of endothelial activation, may show a more dynamic response to vasculoprotective modalities such as del Nido cardioplegia [35]. In contrast, patients undergoing AVR usually have a hypertrophied myocardium due to chronic pressure overload caused by aortic stenosis [10]. Microcirculatory dysfunction and impaired coronary reserve in such circumstances may limit the modulatory effects of cardioplegia on endothelial adhesion molecules, thereby influencing the observable reduction in E-selectin levels despite overall oxidative and inflammatory protection [47].

The primary aim of our study was to move beyond historical clinical comparisons and provide a deeper biomarker assessment of vasculoprotective effects through coronary sinus sampling. We also aimed to clarify the molecular basis by which del Nido may provide endothelium and redox protection during cardiac bypass by concentrating on biomarkers such as E-selectin, endocan, and TNF-α. We believe that these findings reinforce previous evidence of clinical non-inferiority [22] and may serve as a basis for future studies integrating biochemical endpoints with long-term outcomes [36, 48].

Conclusion

Current results indicate that del Nido cardioplegia exhibits significant vasculoprotective properties via modulation of coronary sinus Endocan, E-selectin, and TNF-α-related effects in open-heart surgery. Based on current research, del Nido cardioplegia may offer efficient cardiovascular protection in AVR and CABG surgeries. The degree of cardiovascular protection effectiveness of del Nido may depend on the type of surgery and the different cut-off values of the studied endothelial biomarkers. Statistical analysis, adjusted for baseline (pre-clamp) values, confirmed that del Nido cardioplegia was associated with significantly lower TOS and Endocan levels and reduced TNF-α compared to blood cardioplegia in the CABG group, whereas no significant differences were observed in the AVR group. These findings suggest that del Nido cardioplegia may provide effective cardiovascular protection in both AVR and CABG surgeries, with the degree of protection potentially influenced by the type of surgery and the cut-off values of studied endothelial biomarkers. Moreover, surgery type-specific selection of the cardioplegia that affects the levels of the biomarkers studied may also offer a new strategy to protect against CPB-related endothelial dysfunction and inflammatory conditions. This study’s primary innovation is its assessment of localized endothelial responses via coronary sinus blood sampling and the use of a multiplex biomarker panel, offering research evidence for the coronary vascular endothelial protective effects of del Nido cardioplegia solution.

Abbreviations

AUC

Area under curve

AVR

Aortic valve replacement

CABG

Coronary artery bypass grafting

CAD

Coronary artery disease

CC

Cross-clamp

CPB

Cardiopulmonary bypass

OSI

Oxidative stress index

ROC

Receiver operating characteristic curve

ROS

Reactive oxygen species

SYNTAX score

SYNergy between PCI with TAXUS and Cardiac Surgery score

TAS

Total antioxidant status

TNF-α

Tumor necrosis factor-alpha

TOS

Total oxidant status

Author contributions

Study conception and design: F.K., T.C., B.K.; Patient selection, surgical operations, and blood collection process: F.K., T.C., M.R., B.K.; Integrity of the work among group authors: F.K., T.C.; Optimization of the preanalytical phase: K.B.; Determination of biomarkers: K.B.; Raw data collection: F.K, T.C., Ş.T.; Statistical analysis and preparation of figures: Ş.T.; Analysis and interpretation of results: F.K., T.C., Ş.T., B.K.; Draft manuscript preparation: F.K., T.K., Ş.T., B.K.; Final approval of the scientific quality of the paper: F.K., B.K.

Funding

This study was funded by the Scientific Research Projects Coordination Unit of the University of Health Science, Istanbul (Project number: 2022/109) and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

Code Availability

During the preparation of this work, the authors used Grammarly to improve language and readability. After using this tool/service, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication.

Declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.