Introduction

Coronary artery bypass grafting (CABG) surgery is an effective invasive treatment of ischemic heart diseases. CABG, performed with cardiopulmonary bypass (CPB), provokes a systemic inflammatory response caused by multiple factors, including the surgical trauma itself, CBP, and myocardial ischemia-reperfusion [1]. Inflammation-induced tissue injury predominantly causes subclinical organ dysfunction but produces a clinical effect in patients with an excessive inflammatory response or in patients with limited functional reserve [2]. Ischemia and reperfusion increase the production of reactive oxygen and nitric oxide species [3] and induces oxidative stress. The oxidative stress may precede peak systemic inflammatory response and further contribute to it [4] and is also likely to contribute to secondary CPB-associated postoperative complications [5]. The antioxidant administration may reduce the oxidative stress of CPB [6]. Although improvement in inflammatory markers or patient outcomes has not been detected in some of the antioxidant administration studies [7], other clinical studies have shown that nutrients with antioxidant activity may improve patient outcomes in surgeries applying CPB [8].

Vitamin E is a hydrophobic antioxidant. The concentration of vitamin E may decrease following CABG surgery [9]. Experimental studies have shown that dietary supplementation with vitamin E can improve myocardial tolerance towards ischemia and reperfusion [10]. Perioperative infusion of vitamin E has prevented the postoperative depletion of plasma vitamin E after cardiac surgery. However, its use has not resulted in a measurable reduction in myocardial injury or postoperative clinical outcomes [9]. However, after acute myocardial infarction, which can be associated with ischemia/reperfusion injury, vitamin E and C supplementation had a positive effect on clinical outcomes [11]. In addition to antioxidant vitamins, CABG is associated with a reduction of blood levels of several trace elements, including zinc [12] which probably is mediated by the inflammatory redistribution [13]. Some metalloenzymes with redox capacity are zinc-dependent. Furthermore, zinc can induce metallothioneins, which can scavenge hydroxyl radicals [14]. A protective role of zinc in myocardial recovery from oxidative stress induced by ischemia and reperfusion has been reported [15]. The postoperative decrease in zinc levels may be attenuated by zinc supplementation.

Regarding the possible influence of the antioxidant defense system on the complications after CABG surgery performed with CPB, there seems to be a lack of information on the effects of antioxidant nutrients in CABG surgery, and there is a need for more studies in this area. In the current study, we tested the hypothesis that preoperative high-dose vitamin E and zinc supplementation followed by daily lower doses postoperatively in patients undergoing elective CABG surgery may attenuate the postoperative inflammatory response and improve Systemic Inflammatory Response Syndrome (SIRS) and Sequential Organ Failure Assessment (SOFA) score and, thus, might improve the clinical course of these patients, including intensive care unit (ICU) and postoperative length of stay.

Materials and methods

Patients and study design

This study included patients of both sexes aged 18–80 years who were scheduled to undergo elective coronary artery bypass grafting using the on-pump method at Shafa university hospital (Kerman University of Medical Sciences, Kerman, Iran) between June 2022 and January 2023. Eligible patients were CABG candidate patients who did not have end-stage renal disease, liver cirrhosis, chronic rheumatic diseases, active cancer (under treatment), and severe infection (white blood cells (WBC) > 12,000 mm3) before surgery. Patients who regularly used zinc, vitamin E, vitamin C or multivitamin, or high-dose fish oil (more than 3 g of per week) supplements during the month leading up to surgery were not included in the study. Patients who were regularly taking corticosteroids and non-steroidal anti-inflammatory drugs (except A.S.A) were excluded. Participants were also excluded from the study if they performed off-pump surgery, required re-surgery, and changed their treatment plan.

The study used a double-blind, randomized, placebo-controlled design. A computer-generated randomization protocol (by Random Allocation Software) was used to randomly assign patients in a 1:1 ratio to the intervention group or placebo group. Block randomization was employed and a block size of 4 was used. Each block sequence was placed in a sealed opaque envelope. Patients were allocated to one of the two study groups by the study’s research assistant according to the randomization sequence. Patient allocation to the groups was not known to either the responsible personnel or the investigator. One group of patients received a loading dose of 1200 international units (IU) of vitamin E (3 × 400 IU as alpha-tocopheryl acetate) and 120 mg of zinc (3 × 40 mg as zinc gluconate) orally on the day before surgery which was followed by 200 IU of vitamin E and 30 mg of zinc per day orally from the second postoperative day to three weeks later. The other group received placebo capsules. In addition to study supplements or placebos, all patients received a standard micronutrient mixture consisting of 500 mg of vitamin C, and magnesium (as magnesium sulfate 50%, 8 mL) intravenously, during surgery.

The study was performed at a university hospital (Shafa Hospital, Kerman University of Medical Sciences, Kerman, Iran). The study was in accordance with the Declaration of Helsinki, and the protocol was approved by an ethics committee (The ethics committee of the National Nutrition and Food Technology research institute, Shahid Beheshti University of Medical Sciences). Informed consent was obtained from patients. This trial was registered at ClinicalTrials.gov website (NCT05402826).

Outcome measures

The primary clinical outcome was the ICU length of stay (LOS). The secondary clinical outcomes were SIRS, and SOFA scores on the 3rd day after the operation, duration of intubation in the ICU, and plasma concentrations of inflammatory and biochemical factors. The SOFA score, based on cardiovascular, respiratory, renal, hepatic, neurological, and coagulation systems, was used to evaluate the disease severity of patients in the ICU. SIRS was defined as the presence of at least two of the following criteria: (1) body temperature <36 or >38 °C, (2) respiratory rate >20, (3) heart rate >90, or (4) WBC > 12,000/mm3 or <4000/mm3 or >10% polymorphonuclear leukocytes [16]. Systemic inflammation was quantified through plasma C-reactive protein (CRP) and pentraxin 3 (PTX3), as well as via plasma interferon-gamma (IFN-γ) and interleukin 4 (IL-4). At the same time, a change in redox regulation was assessed via plasma malondialdehyde (MDA).

Blood sample collection and processing

Blood samples were taken preoperatively (day 0) and on postoperative days 3, and 21. Venous blood was collected in heparinized vacutainers, subjected to centrifugation to obtain plasma, and stored at −80 °C for later analyses of inflammation markers, MDA, and biochemical factors.

Plasma albumin, zinc, and CRP concentrations were measured with a commercial kit (Audit Diagnostics, Cork, Ireland) by an autoanalyzer. Plasma PTX3, IFN-γ, and IL-4 were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Biolegend, San Diego, USA) according to the manufacturer’s protocols. Plasma cardiac troponin I (cTnI) was measured via an ELISA kit (Monobind, Inc., Lake Forest, CA, USA). Plasma MDA, an indicator of lipid peroxidation, reacts with thiobarbituric acid at high temperatures and acidic conditions, to generate a pink MDA- thiobarbituric acid adduct that is quantified at an optical density of 532 nm. Plasma MDA was measured after incubation with an oxidizing agent (2 mM CuCl2), or without added CuCl2 [17].

Statistical analysis

The sample size was calculated considering ICU length of stay as the primary outcome variable. Data from a previous study, in which the ICU length of stay was reduced in patients who received oral vitamin E and allopurinol prior to CABG surgery [18], was used for sample size calculation. Considering an α error rate of 5, 95% power, and 20% loss rate, 30 patients needed to be recruited for each study group. Analyses were done per protocol. Data were analyzed by IBM SPSS Statistic 26.0 (IBM Corporation). Data normality was determined by Kolmogorov–Smirnov test. Data are expressed as the means ± standards deviation, median (the first quartile, the third quartile), or as a percentage. Between-group comparisons of baseline values were performed using the chi-square test or independent t-test (or Mann–Whitney U-test for skewed continuous variables). Between-group differences in the primary outcome (length of ICU stay), as well as the SOFA score on the third postoperative day, were determined using univariate analysis of covariance (ANCOVA) adjusted for age, body mass index (BMI), nutritional risk screening (NRS) score, perfusion time during surgery, and baseline blood hemoglobin and WBC. Changes in the plasma level of the studied markers were analyzed by general linear model repeated measures ANCOVA, adjusted for covariates. Univariate ANCOVA adjusted for covariates was used to compare values between groups at postoperative days 3 and 21. Because the distribution of the ICU length of stay, SOFA score, plasma inflammatory factors, MDA, and cTnI were skewed, they were transformed using the natural logarithm to achieve approximate normality. A p-value less than 0.05 was considered statistically significant.

Results

Among the 80 patients who entered, 78 patients completed the study (40 in vitamin E and zinc and 38 in the placebo group) (Fig. 1). The baseline characteristics of patients in both groups are shown in Table 1. The mean age of the study participants was 60.19 ± 9.28 years. Among 78 patients, 27 patients had a history of diabetes, and 52 patients were male. The patients in the placebo group were modestly older. Furthermore, the placebo group tended to have a lower BMI (p = 0.08), as well as lower blood hemoglobin (p = 0.06), and higher circulating WBC (p = 0.07). The perfusion time during surgery was higher in the placebo compared to the intervention group (p = 0.039). There was no operative mortality. The NRS was performed preoperatively to assess nutritional risk. The placebo group tended to have a higher NRS score (Table 1). Oral feeding was started shortly after the end of the intubation period (after extubation).

Figure 1
figure 1

Flowchart of the study patients.

Table 1 Baseline characteristics of study patients.

The ICU LOS tended to be shorter in the intervention group and the hospital LOS was shorter (Table 2). Intubation duration was not different between the two groups. The SOFA score on postoperative day 3 was lower in the intervention group. However, after adjusting for covariates, no significant difference was observed. SIRS (defined as presence ≥2 criteria) on postoperative day 3 was lower in the intervention group than in the placebo group, but the difference was not statistically significant (p = 0.67). During hospitalization, sepsis with positive blood culture was not observed in any of the two group patients. In addition to prophylactic antibiotic administration after surgery, 12 patients in the placebo group and seven patients in the intervention group required additional antibiotherapy for infectious complications, and the difference between the two groups was not significant in this respect (p = 0.148). Thirty-day mortality occurred in two patients in the placebo group whereas no deaths were observed in the vitamin E and zinc group (p = 0.142).

Table 2 Outcome variables in the two studied groups.

Figure 2 shows the changes in plasma concentration of zinc, copper, inflammatory markers, and MDA. The plasma zinc level in the vitamin E and zinc group was significantly higher than the placebo group at both postoperative time points (day 3 and day 21). Repeated measure ANCOVA showed significant differences in plasma CRP, PTX3, and IFN-γ between the two groups. Plasma MDA (incubated with the oxidizing agent) also differed between the two groups (Fig. 2H). Comparison between groups at each time point showed that plasma PTX3 and IFN-γ were lower in the vitamin E and zinc group on both postoperative days 3 and 21, while plasma CRP was lower only on postoperative day 21 and MDA on day 3 after the operation. There was no significant difference between the two groups in the concentration of plasma MDA (incubated without oxidant agent) (Fig. 2G) and the level of IL-4 (Fig. 2F).

Fig. 2: Plasma levels of biomarkers in the two groups before and after the surgery.
figure 2

Data are expressed as the means ± standard deviation. The changes over time (Time) and the treatment effect (group) were analyzed by using repeated measure ANCOVA, adjusted for respective baseline values, age, BMI, NRS score, perfusion time during surgery, baseline blood WBC, and hemoglobin, as covariates. Except for plasma zinc, albumin, and IL-4, logarithmic transformation was performed on other data before analysis. *p < 0.05, **p < 0.01, ***p < 0.0001 corresponding to between-group comparisons at each time point by using univariate ANCOVA, adjusted for respective baseline value and age, BMI, NRS score, perfusion time during surgery, blood WBC and hemoglobin at baseline, as covariates. Data were log-transformed before analysis. CRP C-reactive protein, IFN-γ interferon γ, IL-4 interleukin 4, MDA malondialdehyde, PTX3 pentraxin 3.

In Table 3, plasma concentrations of cTnI, aminotransferases, and blood cells are shown. No significant differences between the two groups were observed regarding these parameters, except for aspartate aminotransferase (AST), whose level in the intervention group was significantly lower than the placebo group on day 21 after the operation.

Table 3 Blood biochemical and hematological factors in the two groups before and on the 3rd and 21st days after the surgery.

Discussion

Our study is among the few studies reporting the effects of perioperative micronutrient supplementation among patients undergoing CABG surgery. Preoperative vitamin E (1200 IU) and zinc ( 120 mg) supplementation in adult patients undergoing CABG surgery, followed by 200 IU vitamin E and 30 mg zinc per day postoperatively, seems to be effective in shortening hospital LOS. These dietary supplements had no significant effect on SIRS and SOFA score on postoperative day 3, but decreased postoperative plasma concentrations of PTX3, CRP, IFN-γ, MDA, and AST, without significant impacts on the postoperative plasma IL-4, cTnI levels.

The motivation for using the combination of vitamin E and zinc supplements in the present study was their antioxidant as well as their immunomodulatory effects. Furthermore, it has previously been shown that plasma zinc [12] and vitamin E [9] are reduced following CABG surgery. This reduction may be a redistribution that occurs as a result of inflammatory responses [19]. In the present study, we did not measure plasma vitamin E levels. Still, the dose of tested vitamin E in the present study is comparable to the amount used in the study by Lassnigg et al., who showed that four doses (270 mg each) of vitamin E between 16 h before and 48 h after CABG surgery normalizes plasma vitamin E levels in the postoperative period [9]. On the other hand, the tested dose of zinc was comparable to the amount used in a previous study, in which patients received 50 mg for the first two days after admission to ICU, followed by 25 mg/day [20]. Although the doses of vitamin E and zinc in the present study were higher than the Dietary Reference Intake values, they were not higher than the Tolerable Upper Intake Levels. Besides, the recommended dietary amounts are recommended for healthy people and the amount required during acute illness may be different. In the case of zinc, studies have shown that the medical doses used are relatively safe, and regardless of dietary intake, the use of medical doses of zinc may be beneficial for a wide variety of clinical situations [21].

Perfusion time was different between the two groups. Since cardiac surgery with CPB induces activation of a systemic inflammatory response [1] and perfusion time has been shown to correlate with postoperative inflammatory mediators levels [22], it was included as one of the confounding factors in statistical analysis.

The ICU and hospital length of stay as the primary outcomes were shorter in the intervention than in the placebo group. This is in contrast to the findings of a previous study in which the administration of several micronutrients with antioxidant activity, including vitamin E and zinc, in patients admitted to the ICU after complex cardiac surgery did not shorten the length of stay in the ICU [20]. This discrepancy could be related to the study population which was complicated heart surgery patients or to the lack of preoperative administration of loading doses of micronutrients. However, our finding is somehow consistent with the result of a previous study in which prophylactic treatment with vitamin E and allopurinol in patients with pre-existing chronic kidney disease reduced the length of stay in the ICU after CABG surgery [23]. Furthermore, the early administration of vitamins E and C reduced ICU length of stay in critically ill surgical patients [24].

The shorter ICU and hospital length of stays might be partly caused by the reduction of inflammation intensity in the zinc and vitamin E groups. Systemic inflammation can predict poor outcomes in patients under elective CABG [25]. Both CRP and PTX3, which are acute-phase proteins, are released significantly during and after CABG surgery. In a study in which serum level of PTX3 was prospectively monitored in ICU patients, higher PTX3 level was associated with sepsis development, and a higher 90-day mortality rate [26]. Furthermore, in patients undergoing cardiac surgery with the use of CBP, the dynamics of serum CRP levels were comparable in patients with SIRS and those with no SIRS, with a peak on the postoperative day 3, whereas serum PTX3 was significantly higher in patients with SIRS than those with uneventful postoperative period [27]. However, perioperative administration of antioxidant vitamins (300 mg/day vitamin E and 500 mg/day vitamin C) in patients who underwent CPB, reduced CRP levels on the first day after surgery compared to the control but, the difference disappeared on the second postoperative day [28]. In addition to antioxidant vitamins, zinc may also be inversely related to oxidative stress and inflammatory factors in critically ill patients, particularly in those with sepsis [29].

In the present study, the lower IFN-γ plasma level on the 3rd and 21st days after surgery in the vitamin E and zinc supplementation group could also be related to the lower intensity of inflammation. Aberrant expression of IFN-γ may be associated with some inflammatory and autoimmune diseases [30] and the decrease in IFN-γ synthesis might prevent excessive stimulation of the non-specific immune system. In a previous study on patients who underwent CABG with CPB, serum IFN-γ levels did not change significantly on the 3rd day after surgery. However, its production by cultured mixed mono-nuclear leukocytes was temporarily decreased on postoperative day 1 but recovered on postoperative day 3 [31]. In another study, mixed mono-nuclear leukocytes of patients who underwent CABG surgery were obtained pre-operatively and on the first and third postoperative days and cultured along with adding exogenous IFN-γ. The addition of IFN-γ significantly increased pro-inflammatory cytokine tumor necrosis factor-α but did not affect the synthesis of anti-inflammatory cytokines interleukin-10, and IL-4 levels [32].

Enhanced lipid peroxidation can lead to the production of end products such as MDA, which, by attaching to biomolecules, can generate self-MDA epitopes capable of inducing the immune system to neutralize, causing inflammatory responses [33]. In the present study, the vitamin E and zinc supplementation reduced the MDA of the plasma that was incubated with oxidant. A significant decrease in MDA was observed only for the plasma samples of the third postoperative day, without a significant effect on the samples of the 21st postoperative day. It seems that the postoperative acute phase responses and the production of free radicals are higher on the third than on the 21st day after the operation. Therefore, one reason for the observed findings could be related to the higher intensity of oxidative stress in the early days after surgery, which vitamin E and zinc were able to reduce it. However, since the spectrophotometric method to detect MDA is less sensitive than fluorometric methods, it may not have revealed the difference on the 21st day.

The plasma cTnI concentration in the intervention group was not significantly different from the placebo group. However, the beneficial effects of antioxidant supplements on cardiac markers have been reported in some studies. In this regard, the administration of vitamin E into the coronary arteries during surgery attenuated the post-surgery increase in cTn-I levels [34]. In the current study, vitamin E and zinc supplementation decreased AST levels on the 21st day after surgery compared to the placebo group. AST is released when cells are damaged. It used to be a regular cardiac marker before more specific markers became common. Elevated postoperative AST level has been suggested as an independent predictor of early and late mortality after CABG [35]. Furthermore, increased pre- and post-operative AST to ALT ratios seem to be associated with an increased incidence of acute kidney injury after elective CABG surgery [36]. The vitamin E supplement consumed was probably effective in reducing AST levels. Vitamin E supplementation has been reported to improve AST levels in patients with liver disease [37].

The present study had some limitations. Due to the diversity of eligibility criteria, the generalizability of the results is limited to relatively low-risk patients undergoing CABG surgery. The single-center nature of this trial can be considered both a limitation and an advantage due to the reduction of variation in care. Furthermore, we did not collect SIRS and SOFA scores on different days after surgery, which might have provided valuable information. Moreover, the concentration of vitamin E in the plasma of the patients was not measured. Finally, despite the inclusion of 78 patients, the study could still be underpowered for some study variables. Despite this, many parameters were oriented towards a clinical benefit in favor of nutrient supplementation. However, a larger trial is needed.

Conclusion

Perioperative supplementation of vitamin E and zinc in CABG surgery patients was associated with postoperative shortened ICU and hospital length of stay and lower postoperative plasma concentrations of inflammatory biomarkers. Although the reduction in ICU length of stay observed following supplementation was not long, it could be considered an achievement with such easy access and cheap intervention. Following cardiac surgery, the plasma concentration of several micronutrients undergoes significant changes, and it appears that this field requires more investigation to reveal their association with some complications after surgery. The optimal combination and dose of micronutrients remain to be determined but could include zinc and vitamin E.