Vitamin C supplementation attenuates oxidative stress and improves erythrocyte deformability in cardiac surgery with cardiopulmonary bypass

Ying-Hsuan Tai; Hsiang-Ling Wu; You-Hsiang Chu; Cheng-Hsiung Huang; Shung-Tai Ho; Tso-Chou Lin; Chih-Cherng Lu

doi:10.4103/0304-4920.358234

ORIGINAL ARTICLE

Year : 2022 | Volume : 65 | Issue : 5 | Page : 241-249

Vitamin C supplementation attenuates oxidative stress and improves erythrocyte deformability in cardiac surgery with cardiopulmonary bypass

Ying-Hsuan Tai¹, Hsiang-Ling Wu², You-Hsiang Chu³, Cheng-Hsiung Huang⁴, Shung-Tai Ho⁵, Tso-Chou Lin⁶, Chih-Cherng Lu⁷
¹ Department of Anesthesiology, Shuang Ho Hospital, Taipei Medical University, New Taipei City; Department of Anesthesiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
² Department of Anesthesiology, Taipei Veterans General Hospital; School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
³ Department of Anesthesiology, Taipei Veterans General Hospital; Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
⁴ School of Medicine, National Yang Ming Chiao Tung University; Division of Cardiovascular Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
⁵ Department of Anesthesiology, Tri-Service General Hospital, National Defense Medical Center, Taipei; Department of Anesthesiology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
⁶ Department of Anesthesiology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
⁷ Department of Anesthesiology, Taipei Veterans General Hospital; Graduate Institute of Life Sciences; Institute of Aerospace Medicine, National Defense Medical Center, Taipei, Taiwan

Date of Submission	20-May-2022
Date of Decision	03-Jul-2022
Date of Acceptance	12-Jul-2022
Date of Web Publication	10-Oct-2022

Correspondence Address:
Prof. Chih-Cherng Lu
Department of Anesthesiology, Taipei Veterans General Hospital, Taipei 112
Taiwan

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0304-4920.358234

Abstract

Cardiopulmonary bypass (CPB) depletes endogenous Vitamin C and generates oxidative stress in cardiac surgery. This study aimed to clarify whether Vitamin C supplementation reduces oxidant production and improves erythrocyte deformability in cardiac surgery with CPB. In a randomized and controlled design, 30 eligible patients undergoing cardiac surgery with hypothermic CPB were equally assigned to the Vitamin C group and control group. Subjects of the Vitamin C group and control group received an intravenous infusion of Vitamin C 20 mg·kg^–1 and a placebo during rewarming period of CPB, respectively. We measured the plasma level of reactive oxygen species (ROS) and phosphorylation levels of non-muscle myosin IIA (NMIIA) in erythrocyte membrane, as an index of erythrocyte deformability, before and after CPB. Vitamin C supplementation attenuated the surge in plasma ROS after CPB, mean 1.661 ± standard deviation 0.801 folds in the Vitamin C group and 2.743 ± 1.802 in the control group. The tyrosine phosphorylation level of NMIIA after CPB was upregulated in the Vitamin C group compared to the control group, 2.159 ± 0.887 folds and 1.384 ± 0.445 (P = 0.0237). In addition, the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and focal adhesion kinase (FAK) in erythrocytes was concurrently enhanced in the Vitamin C group after CPB. The phosphorylation level of endothelial nitric oxide synthase in erythrocytes was significantly increased in the Vitamin C group (1.734 ± 0.371 folds) compared to control group (1.102 ± 0.249; P = 0.0061). Patients receiving Vitamin C had lower intraoperative blood loss and higher systemic vascular resistance after CPB compared to controls. Vitamin C supplementation attenuates oxidative stress and improves erythrocyte deformability via VASP/FAK signaling pathway in erythrocytes during CPB.

Keywords: Antioxidant, ascorbic acid, non-muscle myosin IIA, reactive oxygen species

How to cite this article:
Tai YH, Wu HL, Chu YH, Huang CH, Ho ST, Lin TC, Lu CC. Vitamin C supplementation attenuates oxidative stress and improves erythrocyte deformability in cardiac surgery with cardiopulmonary bypass. Chin J Physiol 2022;65:241-9

How to cite this URL:
Tai YH, Wu HL, Chu YH, Huang CH, Ho ST, Lin TC, Lu CC. Vitamin C supplementation attenuates oxidative stress and improves erythrocyte deformability in cardiac surgery with cardiopulmonary bypass. Chin J Physiol [serial online] 2022 [cited 2023 Nov 30];65:241-9. Available from: https://www.cjphysiology.org/text.asp?2022/65/5/241/358234

Introduction

Cardiopulmonary bypass (CPB) exposes patients to multiple physiologic abnormalities and potentially causes vital organ dysfunction or injury.^[1] This includes hypothermia, hemodilution, microthrombosis, proinflammatory response, and ischemia/reperfusion injury.^[1] The passage of blood through non-endothelial extracorporeal circuit activates neutrophils and generates reactive oxygen species (ROS).^[2] In addition, reperfusion after ischemia during CPB initiates a series of biochemical reactions and produces excessive amount of ROS.^[2] Studies have shown that the altered redox state and oxidative stress of CPB may induce myocardial injury and atrial fibrillation in cardiac surgery.^[3],[4]

Erythrocytes play an important role in physiological homeostasis, including control of redox regulation, blood rheology, and tissue metabolism.^[5],[6] Membrane deformability is an essential feature of erythrocytes and participates in the regulation of microvessel recruitment and tissue oxygen supply.^[7],[8] The mechanical and oxidative stress of CPB may reduce the adaptive response of erythrocytes to shear forces, cause microcirculatory derangements, and thereby induce tissue ischemia.^{[9],[10],[11]} However, there have been few proven treatments for ameliorating the injury of oxidative stress during CPB.^{[12],[13],[14]}

Vitamin C (ascorbic acid) is an essential micronutrient and acts as a potent scavenger for ROS in humans.^[15] Hill et al. recently reported that over half of the patients undergoing cardiac surgery had a suboptimal level of Vitamin C before surgery, which was further depleted by CPB.^[16] Vitamin C supplementation improves endothelial function with stronger benefits in people at higher cardiovascular risk.^[17],[18] Antioxidant therapy with Vitamin C may prevent organ dysfunction following cardiac surgery, including atrial fibrillation and vasoplegic syndrome.^[19],[20] Experimental studies demonstrated that low Vitamin C concentrations in erythrocytes are linked to decreased erythrocyte deformability in diabetic patients.^[21] Vitamin C supplementation maintains the concentration of erythrocytes glutathione and improves the antioxidant capacity of blood in healthy adults.^[22] However, it remains unclear whether Vitamin C therapy reduces oxidative stress and improves erythrocyte deformability in the setting of cardiac surgery with CPB.

This study aimed to evaluate the effect of Vitamin C supplementation on the production of ROS during CPB and to investigate how Vitamin C affects the molecular determinants of cellular deformability and improves erythrocyte deformability among patients undergoing cardiac surgery. Based on the current evidence,^{[15],[16],[17],[18],[19],[20],[21],[22]} we hypothesized that Vitamin C therapy during CPB reduces the plasma level of ROS and enhances the erythrocyte deformability in cardiac surgery.

Materials and Methods

Patient selection criteria

This study was approved by the Institutional Review Board of Taipei Veterans General Hospital, Taipei, Taiwan (approval number: IRB-TPEVGH No. 2019-06-003CCF; date of approval: July 3, 2019) and was conducted in accordance with the standards of the Helsinki Declaration and the institutional regulations on human experimentation. Written informed consent was obtained from each participant. We enrolled patients who underwent cardiac surgery requiring hypothermic CPB at the medical center between April and December 2020. Patients were excluded for the following conditions: age below 20 years old, current use of digitalis, history of hyperuricemia, advanced chronic kidney disease (estimated glomerular filtration rate <30 mL·min⁻¹·1.73 m⁻²), red blood cell disorder (e.g., glucose-6-phosphate dehydrogenase deficiency and thalassemia), and preoperative left ventricular ejection fraction <30%.

Anesthesia and hemodynamic management

Standard monitoring was applied to each patient, including five-lead electrocardiogram, peripheral pulse oximetry, capnography, direct arterial blood pressure, and pulmonary artery catheterization for continuous cardiac output monitoring (Swan-Ganz thermodilution catheter 7.5 Fr, Edwards Lifesciences, CA, USA). Fentanyl 1–2.5 μg·kg⁻¹ and propofol 1–2.5 mg·kg⁻¹ were given for induction of anesthesia. Rocuronium 0.8–1.0 mg·kg⁻¹ was used as the neuromuscular blocking agent to facilitate tracheal intubation. General anesthesia was maintained using sevoflurane 1–3 vol% in oxygen, with a fraction of inspired oxygen of 0.5–1.0.

Patient's volume status was evaluated using stroke volume variation based on arterial pressure waveform analysis (FloTrac/Vigileo, Edwards Lifesciences, Irvine, CA, USA). One hundred and fifty mL of lactated Ringer's solution or sodium chloride solution 0.9% were given if stroke volume variation increased above 15%. Red cell transfusion was considered if serum hemoglobin concentration fell below 8.0 g·dL⁻¹. If the cardiac index (CI) dropped below 2.5 L·min⁻¹·m⁻², we used an intravenous infusion of epinephrine 2–10 μg·min⁻¹ to improve myocardium contractility. An experienced research assistant recorded the measures of hemodynamic parameters 15 min after induction of anesthesia (baseline) and 15 min after weaning from CPB, including heart rate, systemic and pulmonary blood pressure, CI, systemic vascular resistance (SVR), pulmonary vascular resistance (PVR), and central mixed venous oxygen saturation (ScvO₂). Skin blood flow was measured on the palmar surface using a laser Doppler flowmeter (DRT4 Instrument, Moor Instruments, Axminster, UK).

Techniques of cardiopulmonary bypass

CPB was performed using HL-30 roller pumps (Maquet, Rastatt, Germany) and Affinity NT oxygenators (Medtronic, Fridley, MN, USA). The pump flow was adjusted to acquire an output of 2.2 L·m⁻² of body surface area and was reduced to 0.5 L·min⁻¹ for aortic clamping and unclamping. The core temperature was maintained between 32 and 34°C. Antegrade or retrograde cardioplegia was used with 15–29°C Custodiol HTK (Koehler Chemi, Alsbach-Haenlien, Germany) on a case-by-case basis. Weaning from CPB was initiated after systemic temperature was above 36°C.

Vitamin C supplementation

Patients were allocated to the Vitamin C group or control group in a ratio of 1:1 using a random number table for simple randomization. A meta-analysis of randomized controlled trials has shown that supplementation of Vitamin C 2 g before and 1–2 g/day after surgery is safe and effective in preventing atrial fibrillation after cardiac surgery.^[19] Accordingly, we initiated an intravenous infusion of Vitamin C 20 mg·kg⁻¹ administered within 10 min at the rewarming phase of CPB in the Vitamin C group. Patients of the control group received sodium chloride solution 0.9% as the placebo.

Study outcomes

Smith et al. have shown that nonmuscle myosin IIA (NMIIA) in erythrocytes interacts with membrane skeleton to control membrane deformability, and NMIIA phosphorylation is highly correlated with erythrocyte deformability.^[23] In this study, we used the phosphorylation level of NMIIA in erythrocyte membrane as an index of erythrocyte deformability. The primary outcome was the change of tyrosine phosphorylation of NMIIA in erythrocyte membrane before and after CPB. The secondary outcome was the change of plasma ROS level before and after CPB. To clarify the molecular determinants of erythrocyte deformability in response to exogenous Vitamin C, we also measured the changes of phosphorylation levels of vasodilator-stimulated phosphoprotein (VASP) and focal adhesion kinase (FAK) in erythrocyte membrane. In addition, the phosphorylation level of endothelial nitric oxide synthase (eNOS) in erythrocytes was also determined. The parameters of peripheral microcirculation were also evaluated, including skin blood flow, SVR, serum lactate level, and ScvO₂.

Collection of covariates

We used the electronic medical databank to collect the data of patient characteristics,^[24],[25] including demographics, coexisting diseases, cardiovascular medications used within 30 days before surgery, preoperative left ventricle ejection fraction, preoperative estimated glomerular filtration rate calculated by the Cockcroft–Gault equation,^[26] and European System for Cardiac Operative Risk Evaluation (EuroSCORE) II.^[27] Clinical covariates were types of surgery, anesthesia duration, bypass duration, aortic clamp-cross duration, intraoperative fluid therapy, vasoactive agents, volume of blood loss, and allogeneic blood transfusion.^[28],[29]

Measurement of plasma reactive oxygen species level

We collected patients' peripheral blood at 15 min after induction of anesthesia and at 15 min after discontinuation of CPB for ROS assay. Plasma was centrifuged by 10,000 g at 4°C for 5 min. The supernatant was used for the quantification of ROS levels, performed using a commercial DCF ROS/RNS Assay Kit (Ab238535, Abcam Ltd., Cambridge, UK). Fifty μL of sample were added to the wells of 96-well plate for fluorescence measurement. 50 μL of catalyst was added to each well and incubated at room temperature for 5 min. After mixed well, 100 μL of DCFH solution was added to each well, which was then covered with an adhesive strip and incubated at room temperature for 30 min. We determined the fluorescence with a microplate reader at 480 nm excitation/530 nm emission.

Extraction of erythrocyte membrane protein

Peripheral blood 10 mL was collected through arterial catheters 15 min after induction of anesthesia (baseline) and 15 min after weaning from CPB and was stored in K₂ ethylenediaminetetraacetic acid-containing tubes (Becton Dickinson, NJ, USA). Erythrocytes were isolated using Ficoll Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The extraction procedures of erythrocyte membrane protein were detailed in the authors' prior articles.^{[30],[31],[32]}

Phosphorylation assay of erythrocyte membrane protein

We used immunoprecipitation assay of western blotting to determine the levels of phosphorylation of NMIIA. Protein G Immunoprecipitation Kit (IP-50, Sigma-Aldrich, MO, USA) was utilized to interact with erythrocyte lysate overnight. Antibodies used in this process were purchased from ab55456 (Abcam Ltd., Cambridge, UK) for NMIIA. We used anti-phosphotyrosine antibody, clone 4G10, purchased from 05 to 321 (Millipore, MA, USA), to measure the tyrosine phosphorylation level in immunoblots. Anti-phospho-VASP (Ser²³⁹)/anti-VASP (ab194747 and ab109321, Abcam Ltd., Cambridge, UK) and anti-phospho-FAK (Tyr³⁹⁷)/anti-FAK (#8556 and #3285, Cell Signaling Technology, MA, USA) were used as primary antibodies to detect the phosphorylated and total proteins of VASP and FAK. Specific antibodies for detecting total and phosphorylated eNOS proteins were used, including anti-phospho-eNOS (Ser¹¹⁷⁷)/anti-eNOS (GTX129058/GTX129113, GeneTex Inc., CA, USA) as primary antibodies. Antibodies for β-actin (sc-47778, Santa Cruz Biotechnology Inc., CA, USA) were diluted 1:20,000 in 3% bovine serum albumin. Specific bands by immunoblotting reaction were visualized using the enhanced chemiluminescence system (Millipore, MA, USA). The intensity of blot band was quantified through the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

At least 10 subjects in each group were required to attain a power of 0.8 assuming an alpha level of 0.05 and an effect size of 1.0 in the change of NMIIA phosphorylation level by paired t-test.^[33] Shapiro–Wilk tests and Anderson–Darling tests were used as normality tests. Normally distributed data were presented as mean ± standard deviation (SD). Nonnormally distributed variables were expressed as median with interquartile range (IQR). We compared the distribution of patient and clinical characteristics between the Vitamin C and control groups using either independent t-tests or Mann–Whitney U-tests for continuous variables and Fisher's exact tests for categorical variables, as appropriate. Paired t-test or Wilcoxon signed-rank tests were used to calculate the statistical difference in the changes of NMIIA, VASP, FAK, and eNOS phosphorylation levels, hemodynamic and biochemical parameters before and after CPB, as appropriate. Independent t-test was used to compare two groups at the same time point for western blotting data. We considered P < 0.05 statistically significant for a two-sided test. Statistical analyses were performed using SAS software, version 9.4 (SAS Institute Inc., Cary, NC, USA). Graphs were presented with Prism, version 5.00 (GraphPad Software, San Diego, CA, USA).

Results

Baseline patient characteristics

[Table 1] shows the patient characteristics of the Vitamin C and control groups. There was no difference in the distributions of baseline patient characteristics and types of surgery between the two groups. Regarding intraoperative parameters, there was no difference in anesthesia duration, bypass duration, aortic clamp-cross duration, and the use of blood transfusion or vasoactive agents between groups, either. However, there was a borderline association between Vitamin C supplementation and lower intraoperative blood loss, median 300 mL (IQR: 250–500) and 470 (360–650; P = 0.0585) in the Vitamin C group and control group, respectively.

Table 1: Patient characteristics and surgical and anesthetic management

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Hemodynamic and biochemical data

Both the Vitamin C and control groups had decreased systemic blood pressure, SVR, PVR, serum hemoglobin level, and peripheral skin blood flow after weaning from CPB compared to baseline. Conversely, serum glucose and lactate levels increased after CPB compared to baseline. Compared with control group, the Vitamin C group had significantly higher SVR after weaning from CPB, 750 dyne·sec^–1·cm^–5 (IQR: 690–776) and 680 (670–701), respectively. There was no difference in the post-CPB PVR, skin blood flow, serum lactate level, or ScvO₂ between groups [Table 2].

Table 2: Baseline and postcardiopulmonary bypass hemodynamic and biochemical data

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Plasma reactive oxygen species level

Vitamin C supplementation attenuated the increase in plasma ROS levels after CPB, mean 1.661 ± SD 0.801 folds (P = 0.0283) in the Vitamin C group and 2.743 ± 1.802 folds (P = 0.0136) in the control group. There was no significant difference in the post-CPB plasma ROS level between groups (P = 0.1073) [Figure 1].

Figure 1: Plasma levels of ROS before and after CPB. (*P < 0.05 indicates a significant difference between post-CPB and baseline values by paired t-test). CPB: Cardiopulmonary bypass, ROS: Reactive oxygen species.

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Phosphorylation of non-muscle myosin IIA in erythrocytes

To investigate whether the reduction in plasma ROS levels affected erythrocyte deformability, we measured the phosphorylation level of NMIIA in erythrocytes. NMIIA tyrosine phosphorylation was significantly increased after CPB both in the Vitamin C group (2.159 ± 0.887 folds; P = 0.0025) and control group (1.384 ± 0.445 folds; P = 0.0231) [Figure 2]a. Notably, the tyrosine phosphorylation of NMIIA after CPB was significantly higher in the Vitamin C group than control group (P = 0.0237) [Figure 2]b.

Figure 2: NMIIA tyrosine phosphorylation in erythrocytes. (a) A representative gel was shown. (b) NMIIA tyrosine phosphorylation in erythrocytes was recognized. The level of NMIIA tyrosine phosphorylation after CPB was significantly higher in the Vitamin C group compared to control group. (*P < 0.05 and **P < 0.01 indicate a significant difference between post-CPB and baseline values by paired t-test; ^†P < 0.05 indicates a significant difference between the Vitamin C and control groups by independent t-test). CPB: Cardiopulmonary bypass, NMIIA: Non-muscle myosin IIA.

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Activation of vasodilator-stimulated phosphoprotein and focal adhesion kinase in erythrocytes

The enhanced NMIIA phosphorylation could be induced by changes in structure proteins of downstream signaling molecules in erythrocytes. Therefore, we examined VASP and FAK phosphorylation. VASP phosphorylation (Ser²³⁹) did not significantly change after CPB in the Vitamin C group (1.219 ± 0.398 folds; P = 0.1642) [Figure 3]a. However, VASP phosphorylation of the control group decreased after CPB compared to baseline (0.737 ± 0.166 fold; P = 0.0028). FAK phosphorylation (Tyr³⁹⁷) was significantly increased after CPB in the Vitamin C group (1.214 ± 0.188 folds; P = 0.0146) but not in the control group (0.885 ± 0.156 fold; P = 0.0760) compared with baseline. VASP and FAK phosphorylation after CPB were both significantly higher in the Vitamin C group compared to control group (P = 0.0069 and 0.0019, respectively) [Figure 3]b and [Figure 3]c.

Figure 3: Phosphorylation of VASP and FAK in erythrocytes. (a) A representative gel was shown. (b) VASP serine phosphorylation in erythrocyte membrane under two conditions were recognized. The level of VASP serine phosphorylation after CPB was slightly increased in the Vitamin C group and significantly reduced in the control group. (c) The level of FAK tyrosine phosphorylation after CPB was significantly increased in the Vitamin C group and slightly decreased in the control group. (*P < 0.05 and **P < 0.01 indicate a significant difference between post-CPB and baseline values by paired t-test; ^††P < 0.01 indicates a significant difference between the Vitamin C and control groups by independent t-test). VASP: Vasodilator-stimulated phosphoprotein, FAK: Focal adhesion kinase, CPB: Cardiopulmonary bypass.

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Phosphorylation of endothelial nitric oxide synthase in erythrocytes

Phosphorylation of eNOS in erythrocyte was significantly increased after CPB in the Vitamin C group (1.734 ± 0.371 folds; P = 0.0300) but not in the control group (1.102 ± 0.249 folds; P = 0.0862) compared to baseline. In addition, eNOS phosphorylation level of the Vitamin C group after CPB was significantly higher than that of control group (P = 0.0061) [Figure 4].

Figure 4: Phosphorylation of eNOS in erythrocytes. (a) A representative gel was shown. (b) eNOS phosphorylation in erythrocyte membrane was recognized. The level of eNOS phosphorylation after CPB was significantly higher in the Vitamin C group compared to control group. (*P < 0.05 indicates a significant difference between post-CPB and baseline values by paired t-test; ^††P < 0.01 indicates a significant difference between the Vitamin C and control groups by independent t-test). eNOS: Endothelial nitric oxide synthase, CPB: Cardiopulmonary bypass.

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Discussion

Our study demonstrated that Vitamin C supplementation administered during rewarming of CPB reduced the production of ROS and upregulated tyrosine phosphorylation of NMIIA with concurrent activations of VASP and FAK phosphorylation in erythrocytes among patients undergoing cardiac surgery. The eNOS phosphorylation in erythrocytes was also enhanced by exogenous Vitamin C. In addition, patients receiving Vitamin C had lower surgical blood loss and higher SVR after weaning from CPB compared to controls. The potential benefits of Vitamin C in attenuating oxidative stress and improving erythrocyte deformability may provide an implication for protecting patients from oxidative damage, inflammatory response, and hemolysis. These findings also highlight the important role of erythrocytes in control of redox regulation and vascular tone during extracorporeal circulation.

An in vivo experiment has shown that tyrosine phosphorylation of erythrocyte NMIIA interacts with skeleton proteins and participates in regulation of membrane tension, biconcave disk shape, and deformability.^[23] Our results demonstrated that an intravenous loading of Vitamin C during CPB enhanced the phosphorylation of NMIIA and related structural proteins in erythrocyte membrane, indicating that erythrocyte deformability was preserved. Importantly, erythrocyte deformability plays a key role in regulating microcirculatory blood flow, and reduced erythrocyte deformability independently predicts poor outcomes in cardiovascular disease and sepsis.^[34],[35] To our knowledge, our study is the first to report the potential benefit of Vitamin C in improving erythrocyte health in cardiac surgery. This finding implicates intraoperative supplementation of Vitamin C as a potential therapeutic strategy to reduce the injury of hemolysis and its adverse impact on tissue perfusion during CPB. Ischemia and reperfusion injury is associated with increased oxidative stress and systemic inflammation in patients undergoing CPB.^[1],[2] Accumulating evidence has shown that antioxidant therapy of high-dose Vitamin C is effective in ameliorating oxidative damage related to ischemia and reperfusion.^[36] Furthermore, clinical trials have also reported the potential benefits of Vitamin C supplementation in shortening length of intensive care unit stay and duration of mechanical ventilation, improving myocardium contractility, and preventing pulmonary complications after cardiac surgery.^{[37],[38],[39]}

Interestingly, our results suggested that Vitamin C supplementation was associated with lower volume of intraoperative blood loss and higher SVR after CPB. These findings might be mediated by the following mechanisms. First, Vitamin C is effective in ensuring intact endothelium, especially for people at high cardiovascular risk.^[17],[18] Healthy endothelium may prevent thrombosis formation and platelet consumption, which thereby reduced surgical blood loss. Second, the attenuated oxidative stress after Vitamin C treatment might decrease the inflammatory response and protect patients against vital organ dysfunction, which has been demonstrated in severe sepsis.^[40] A randomized trial recently demonstrated that high-dose intravenous Vitamin C (1.5 g every 6 h) may accelerate the resolution of vasoplegia after cardiac surgery although their finding did not reach statistical significance.^[20] Besides, we found that Vitamin C may improve erythrocyte deformability after CPB, but peripheral skin blood flow did not significantly change. We speculated that the detrimental effect of CPB on peripheral perfusion overwhelmed the protective effect of Vitamin C.

Although our study along with previous studies demonstrated potential benefits of Vitamin C in decreasing oxidative stress and improving perioperative outcome in cardiac surgery, the effective regimen and timing of Vitamin C therapy remain undetermined. In this study, we gave an intravenous loading of Vitamin C 20 mg·kg^-1 within 10 min at rewarming period of CPB, when the stress response generally peaks, to compensate for the dilution effect of extracorporeal circulation. Other studies reported that the regimen of Vitamin C 2–4 g before and 12 g/day for 4–5 days after surgery is effective in preventing atrial fibrillation and shortening length of intensive care unit stay after cardiac surgery.^[19],[37] Another study claimed that an intravenous loading of Vitamin C 1 g 10 min after induction of anesthesia, 10 min before cardiac reanimation, and at sternal closure may reduce the risk of postoperative pulmonary complications.^[39] Future studies should elucidate the optimal dose and timing of Vitamin C supplementation pertinent to important clinical outcomes in cardiac surgery.

In this study, we found that Vitamin C therapy significantly increased the enzyme activity of eNOS in erythrocytes after CPB. Preclinical studies have shown that Vitamin C treatment improved the vascular endothelial function in aortas through preserving tetrahydrobiopterin, restoring eNOS activity, and regulating NAD(P)H oxidase activity in mice.^[41],[42] Our results suggested that circulating erythrocytes may interact with exogenous Vitamin C and participate in the regulation of oxidative stress and vascular endothelial function in surgical injury and extracorporeal circulation. Further studies are needed to clarify the role of erythrocyte eNOS in the control of redox regulation and erythrocyte membrane deformability.

There are some limitations in this study. First, we did not measure the plasma level of Vitamin C in the enrolled patients. Therefore, the therapeutic range of Vitamin C is unknown. Hill and colleagues reported that Vitamin C levels reduced significantly from 6.5 mg·L^–1 before surgery to 2.8 mg·L^–1 48 h after surgery, and the length of intensive care unit stay was shorter in patients with a preoperative level of Vitamin C ≥9 mg·L^–1 than the counterparts.^[16] Ballmer et al. demonstrated that the depletion of plasma Vitamin C might persist for 2 weeks after coronary bypass operations.^[43] It is crucial to determine the therapeutic plasma level of Vitamin C for important patient outcomes. Second, the Vitamin C therapy was only given during CPB, and it remains unclear whether the antioxidative effect could persist through the postoperative period. More studies are needed to determine the effective and safe regimen of Vitamin C therapy after cardiac surgery. Third, we did not evaluate postoperative clinical outcomes, such as length of hospital stay and surgical complications. Therefore, it is uncertain whether the biological effect of Vitamin C could be translated into patient prognosis. Fourth, we did not examine markers of hemolysis and could not evaluate the therapeutic benefits of Vitamin C in erythrocyte membrane. Finally, our sample size was only modest and might be underpowered for some selected outcomes.

Conclusion

An intravenous loading of Vitamin C at rewarming phase of CPB activated tyrosine phosphorylation of NMIIA in the membrane of circulating erythrocytes through VASP/FAK signaling pathway. This result indicated the potential benefits of Vitamin C supplementation in improving erythrocyte deformability in the setting of cardiac surgery. In addition, antioxidant therapy of Vitamin C reduced the oxidative stress from CPB-related ischemia/reperfusion injury and improved the enzyme activity of eNOS. More studies are warranted to quantify the optimal effective dose of Vitamin C and to examine the practical effect of Vitamin C on hemolysis and complications, microcirculatory parameters, and vital organ functions.

Acknowledgments

We are grateful to the staff of Division of Cardiovascular Surgery, Department of Surgery, Taipei Veterans General Hospital, for their assistance and support in this research.

Financial support and sponsorship

This work was supported by the grants from the Ministry of Science and Technology (MOST109-2314-B-038-024), Taipei, Taiwan, Taipei Medical University (TMU110-AE1-B11), Taipei, Taiwan, and Taipei Veterans General Hospital (V109C-050), Taipei, Taiwan.

Conflicts of interest

There are no conflicts of interest.

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