Influence of intravenous vitamin E supplementation in cardiac surgery on oxidative stress: a double-blinded, randomized, controlled study

A. Lassnigg*,1, A. Punz2, R. Barker1, P. Keznickl1, N. Manhart2, E. Roth2 and M. Hiesmayr1

1 Department of Cardiothoracic Anaesthesia and Intensive Care Medicine and 2 Department of Surgical Research, University Hospital of Vienna, Waehringer Guertel 18–20, A-1090 Wien, Austria

Corresponding author. E-mail: andrea.lassnigg@univie.ac.at

Accepted for publication: October 9, 2002


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. I.V. infusions of vitamin E emulsion (all-rac-{alpha}-tocopherol) may reduce ischaemia–reperfusion injury after elective cardiac surgery.

Methods. Forty patients participated in a prospective, double-blind, placebo-controlled, randomized trial, receiving either placebo or four doses (270 mg each) of all-rac-{alpha}-tocopherol between 16 h before and 48 h after surgery. We determined plasma concentrations of vitamin E, vitamin C, malondialdehyde, creatine kinase, troponin I and interleukin 6 and other measures of clinical outcome.

Results. Infusion of vitamin E caused normalization of vitamin E plasma concentrations during and after surgery, but had no effect on the early increase in malondialdehyde concentration or the decreases in antioxidative capacity and the water-soluble antioxidant vitamin C.

Conclusions. Normalization of plasma vitamin E concentrations with parenteral vitamin E emulsion does not affect biochemical markers of myocardial injury and does not affect clinical outcome after cardiac surgery.

Br J Anaesth 2003; 90: 148–54

Keywords: heart, ischaemia; surgery, cardiovascular


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cardiac surgery involves a period of myocardial ischaemia and subsequent reperfusion. This stimulates the formation of reactive oxygen species (ROS)1 2 and can injure the myocardium and impair myocardial recovery.35 Because of their short half-lives, ROS are difficult to measure clinically. Secondary reactive products, such as malondialdehyde, and the systemic depletion of antioxidants, such as bilirubin and vitamins E and C, can be used to quantify oxidative stress.

Vitamin E belongs to the most powerful group of lipid-soluble chain-breaking antioxidants that prevent lipid peroxidation and disruption of membrane integrity.6 Past clinical studies with oral vitamin E are contradictory and have not confirmed the promising results of animal experiments.79 Moreover, in acute clinical conditions, oral administration of vitamin E is not effective because a period of 14 days is needed for a sufficient increase in endogenous vitamin E concentration. Therefore, for high-risk patients a parenteral formulation would be advantageous.

We assessed a specially designed vitamin E suspension (all-rac-{alpha}-tocopherol) for parenteral administration, using biochemical measures of ischaemia–reperfusion injury and other measures of clinical outcome.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patients
Written informed consent was obtained from each patient before inclusion. The study was approved by the ethics committee of the Medical Faculty of the University of Vienna. We considered 58 adult patients about to have elective coronary artery bypass graft surgery, valve surgery or combined procedures (Fig. 1). After randomization into two groups, 23 patients received all-rac-{alpha}-tocopherol (vitamin E group) and 24 received placebo (placebo group). Seven patients were eliminated from further study because of procedure violations. In four patients, the time between the first and second infusions was exceeded because surgery was delayed by emergency operations. In three patients, no cardiopulmonary bypass was used during surgery. One of these patients (placebo group) died of multiple organ failure on the 18th postoperative day. Patients with any of the following conditions or treatments were excluded: emergency cardiac surgery, organ transplantation, angiography within 24 h of the start of the study, impaired renal function (creatinine >2.0 mg dl–1), impaired liver function (serum glutamic-oxaloacetic transaminase >50 U litre–1 and/or serum glutamic-pyruvic transaminase >50 U litre–1), vitamin E substitution, or any other antioxidant therapy within the previous 4 weeks. There were no other restrictions on concomitant medication.



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Fig 1 Trial profile.

 
Dose-finding/pilot study, preparation and administration of all-rac-{alpha}-tocopherol
The term ‘vitamin E’ characterizes a group of lipid-soluble tocopherols and tocotrienoles that have alpha ({alpha}), beta (ß), gamma ({gamma}) and delta ({delta}) isomers. {alpha}-Tocopherol itself has several subforms, the reference being (R,R,R)-{alpha}-tocopherol, with a biological potency of 100%, equal to 1.49 United States Pharmacopoeia (USP) per milligram of substance; 1 USP of vitamin E corresponds to 1 mg of all-rac-{alpha}-tocopheryl acetate. All-rac-{alpha}-tocopherol is a synthetic form consisting of {alpha} isomers and has a potency of 74% of that of (R,R,R)-{alpha}-tocopherol, equal to 1.10 USP mg–1.

We conducted a pilot study with 33 patients to determine the decrease in vitamin E and vitamin C plasma concentrations throughout cardiac surgery with cardiopulmonary bypass. Vitamin E decreased from 8.1 to 5.4 mg litre–1 and vitamin C decreased from 9.7 to 3.9 mg litre–1. On the basis of a normal vitamin E concentration between 6.9 and 15.5 mg litre–1 in humans and according to a pharmacokinetic study in 12 healthy volunteers, a dose of all-rac-{alpha}-tocopherol 270 mg was considered sufficient to maintain normal vitamin E concentrations during surgery.

After giving one all-rac-{alpha}-tocopherol emulsion dose, vitamin E concentrations increased from 11 to 39 mg litre–1 in plasma and from 3 to 4.9 mg litre–1 in red blood cells.

All-rac-{alpha}-tocopherol was given as 50 ml of an oil-in-water emulsion containing all-rac-{alpha}-tocopherol 270 mg, soybean oil 1.25 g, egg lecithin E80 150 mg, glycerin 1.25 g and oleic acid adjusted to pH 8, 10 mg (Fresenius Kabi, Graz, Austria). The placebo was the same emulsion without all-rac-{alpha}-tocopherol, with identical appearance.

Four i.v. infusions were given at the following times: 16–12 h before the start of surgery, immediately before surgery, 20–24 h after the start of surgery and 44–48 h after the start of surgery. A time range of 4 h for each dose was allowed on the basis of the pharmacokinetic study in 12 healthy volunteers to fit with the routine ward procedures. The infusion was given over a period of up to 30 min.

Biochemical measurements
Blood samples were taken at the following times: baseline, before administration of the second infusion, 30 min after reperfusion and before the third and fourth infusions. An additional sample was taken on day 6 after surgery.

Vitamin E and vitamin C
Concentrations of vitamin E and vitamin C in plasma were determined by high-performance liquid chromatography (HPLC). Briefly, for determination of vitamin E, 250 µl EDTA–plasma samples were deproteinized with ethanol and extracted in n-hexane. After evaporation to dryness under nitrogen, the residue was dissolved completely in ethanol–dioxin (1:1) and acetonitrile. An aliquot of the sample extract was injected onto an ODS Hypersil Elite 4 µm, 3x150 mm ID column connected to an HPLC apparatus (Beckman, Fullerton, CA, USA). {alpha}-Tocopherol was detected at 295 nm. The calibration of the overall analytical procedure gave a linear signal (r=0.999) over the ±{alpha}-tocopherol concentration range of 5–40 µM. The coefficient of variation was independent of the concentration and varied in within-day and between-day measurements from 1.4 to 5.3%.

For vitamin C determination, plasma was immediately diluted with 10% (wt/wt) metaphosphoric acid and measured according to the method of Esteve and colleagues.10

Laboratory and clinical variables
Laboratory values determined were malondialdehyde (MDA), creatine kinase, creatine kinase–MB (CK-MB), troponin I, interleukin 6, protein S100B, cholesterol, triglycerides, albumin, uric acid and bilirubin.

Plasma malondialdehyde was assessed by the thiobarbituric acid reaction, according to the method of Wong and colleagues.11 Briefly, plasma was mixed with thiobarbituric acid and boiled with diluted phosphoric acid. The adduct formed was neutralized with sodium hydroxide and deproteinized with methanol. After centrifugation, the supernatant was injected into a Spherisorb C18 RP 5 µm, 4.6x250 mm column. The detection was carried out fluorometrically with excitation wavelength 525 nm and emission wavelength 550 nm.

Because cardiopulmonary bypass can cause ischaemia– reperfusion of the cerebral circulation, we also measured the release of protein S100B into the plasma as an indicator of cerebral cell damage.

Calculation of plasma total antioxidant status (cTAS) was determined with the following equation:12 13

cTAS (mmol litre–1)=(0.63x[albumin])+(1.02x[uric acid])+(1.5x[bilirubin])

The following clinical outcomes were recorded: occurrence of peri- and postoperative myocardial infarction (defined as Q-wave infarction and non-Q-wave infarction associated with substantial enzyme release: CK-MB >50 U litre–1); arrhythmias needing treatment (atrial fibrillation, atrial flutter, ventricular tachycardia, atrioventricular and intraventricular conduction defects); need for postoperative inotropic support and maximal and cumulative doses of five major inotropic drugs (treatment in the operating room and in the intensive care unit was determined by standard treatment plans); use of an intra-aortic balloon pump or a ventricular assist device; duration of stay in the intensive care unit; total duration of hospital stay; and mortality within 30 days.

Statistics
Statistical analysis was by repeated-measures analysis of variance (ANOVA) with planned contrasts, using a commercial package (NCSS 97; NCSS Statistical Software, Kaysville, UT; SAS 6.12, 1996; SAS, Cary, NC, USA). The factors were treatment and time as the repeated measures, and the interaction between treatment and time. A significant interaction term implied a treatment effect. The power was 0.8 at {alpha}=0.05 to detect a change in a biochemical marker of one standard deviation, which corresponds to a 20% difference in MDA concentration. When the requirements for a parametric group comparison were not met, the corresponding non-parametric test was conducted. For patient characteristics, the {chi}2 test was used for categorical data and the Mann–Whitney test for continuous data. Data are expressed as mean (SD). A P-value <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Forty patients—20 in the placebo group and 20 in the vitamin E group—completed the study. No differences in patient characteristics and clinical outcome variables were observed between the two groups (Tables 1 and 2).


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Table 1 Patients’ preoperative characteristics. Values are mean (SD)
 

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Table 2 Patients’ perioperative characteristics. Values are mean (SD). None of the variables were significantly different
 
In the placebo group, five patients had severe perioperative complications (postoperative surgical bleeding and paresis of the right arm, acute renal failure, ventricular fibrillation after myocardial ischaemia immediately after cardiopulmonary bypass, poor wound healing). In the vitamin E group, three patients had severe perioperative complications (transitory ischaemic attack, pacemaker implantation by cardiac asystole, poor wound healing).

Vitamin concentrations
Vitamin E concentrations were not different at baseline between the two groups [placebo group, 10.6 (2.5) mg litre–1; vitamin E group, 9.7 (3) mg litre–1] (Fig. 2). In the placebo group, plasma vitamin E concentration decreased significantly 30 min after reperfusion [5.3 (1.3) mg litre–1; P<0.001] and remained at this low concentration until day 2 after surgery; on postoperative day 6, plasma vitamin E concentration returned to 7.5 (1.6) mg litre–1. In contrast, perioperative vitamin E infusion maintained plasma vitamin E at baseline concentrations during the entire investigation period despite a similar decrease of 30–40% 30 min after reperfusion. Thirty minutes, 1 day and 2 days after reperfusion, vitamin E concentrations were twice as great in vitamin E-supplemented patients than in the placebo group (P<0.001; Fig. 2). This increase in plasma vitamin E concentration in the treatment group was not caused by changes in lipid profile, because correction for cholesterol and triglycerides showed the same results (data not shown).



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Fig 2 Changes in plasma concentration of vitamins E and C for all patients in the treatment and placebo groups at the following times: baseline; before operation (pre); 30 min after aortic declamping (AOX); and days 1, 2 and 6 after surgery. Each blood sample was taken before the vitamin E emulsion was given. Data are mean and SD (bars). For vitamin E, P<0.0001 (repeated-measures ANOVA) for influence of medication, time and interaction; for vitamin C, P=0.65 for medication, P<0.0001 for time and P=0.96 for interaction.

 
Plasma vitamin C concentrations had the same pattern in both groups. Thirty minutes after reperfusion, plasma vitamin C concentration decreased sharply by 50% (P<0.001) in both groups, and remained at the lower concentration throughout the investigation period (Fig. 2).

Plasma calculated total antioxidant status
The course of plasma cTAS was similar to that of vitamin C. On postoperative day 1, cTAS decreased significantly by 35% (P<0.05) in both groups and remained at the reduced concentration until day 6 after surgery (Fig. 3).



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Fig 3 Changes in plasma calculated total antioxidant status (cTAS) for all patients in the treatment and placebo groups at baseline, before operation (pre) and days 1, 2 and 6 after surgery. Each blood sample was taken before vitamin E emulsion was given. Bars indicate SD. P=1.0 (repeated-measures ANOVA) for influence of medication, P<0.0001 for time, and P=0.78 for interaction.

 
Malondialdehyde
MDA plasma concentration changed significantly over time (P<0.0001, repeated-measures ANOVA) but did not differ between the two groups. Thirty minutes after reperfusion, plasma MDA concentration increased by 55% (P<0.05); thereafter it returned to the preoperative value (Table 3).


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Table 3 Laboratory values. Values are mean (SD). *MDA plasma concentration differed significantly over time (P<0.0001, repeated-measures ANOVA). Thirty minutes after reperfusion, plasma MDA concentration increased by 55% (P<0.05)
 
All other laboratory and clinical measures were similar in the two groups (Tables 2 and 3; haemodynamic measurements, Table 4). Inotropic support was necessary in a similar proportion of patients, and the cumulative dose and the duration of use were also similar (data not shown).


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Table 4 Haemodynamic measurements. Values are mean (SD)
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ours is the first study to assess multiple parenteral vitamin E infusions in patients undergoing elective cardiac surgery. Despite measuring several biochemical markers of ischaemia–reperfusion injury, we did not find a protective effect of vitamin E supplementation. Neither did we detect any differences in clinical variables. Possible reasons for the negative findings in this study may be (i) an inadequate supply of vitamin E, (ii) insufficient incorporation of vitamin E into the target cells, and (iii) the lack of efficiency of vitamin E supply when not administered together with other antioxidants, such as vitamin C.

In our study, plasma vitamin E concentrations in the placebo group decreased from 10.6 to a minimum of 4.4 mg litre–1 on the first postoperative day and remained low until day 6. In contrast, in the vitamin E group, vitamin E concentrations were above baseline throughout the study period. The decrease in vitamin C and E concentrations, the increase in MDA and the decrease in antioxidant status began immediately after reperfusion, indicating that oxidative stress had been present. A potential limitation of our study is the fact that the depletion of the endogenous, non-enzymatic hydrophilic defence against oxidative stress was only calculated. We chose this method (an adaptation by Bonnefont-Rousselot and colleagues12 of a method described initially by Miller and colleagues13), because robust biochemical markers could be used. Direct measurement of ROS has been proposed but has not always shown reliable results.2 14 15 Measurement of ROS during bypass operations is complicated by both the transient nature of these species and the complexity of the procedure itself.

We did not measure tissue concentrations of vitamin E, but previous studies have demonstrated that parenteral vitamin E is directly available to endothelial cells in rats.16 Similarly, Chan and Tran17 proved that in vitro incubation of human endothelial cells with vitamin E for 8 h caused vitamin E enrichment within the cells. Therefore, we assumed that a preoperative infusion period of 12 h at the dosage used would return postoperative vitamin E concentrations in plasma to normal and would also be sufficient to increase intracellular vitamin E to an appropriate concentration. Therefore, we believe that the failure to improve clinical outcome after cardiac surgery was not dependent on the route and dosage of vitamin E supply.

Results of the clinical studies of oral supplementation with vitamin E are rather contradictory. Yau and colleagues18 showed that oral administration of vitamin E had a small but significant metabolic and functional effect after elective coronary bypass operations in low-risk patients. Sisto and colleagues19 supplied vitamin E orally for 28 days or, if the patients were at higher risk, for 2 days. Although this short-term oral vitamin E supply was not able to maintain postoperative vitamin E concentrations, this group had a reduced number of perioperative ischaemic events, myocardial infarctions and arrhythmias. However, because the study group also received vitamin C and allopurinol, the demonstrated effect may not have been directly related to the administration of vitamin E.

In another clinical study, pretreating patients undergoing cardiopulmonary bypass with oral vitamin E for 7–10 days and oral vitamin C for 12 h prevented the depletion of vitamin E in plasma but provided no measurable reduction in myocardial injury after operation.20 Mickle and colleagues21 reported that oral vitamin E in three different doses the night before cardiac surgery did not increase myocardial vitamin E concentrations; a 3-day course also had no effect. They concluded that at least 300 mg of vitamin E must be taken orally for 14 consecutive days to double the myocardial vitamin E concentration.

Several studies have shown that chain-breaking efficiency is greatest with coadministration of vitamin E and vitamin C.19 2225 Experimental and clinical studies proved that myocardial oxidative stress is accompanied by lowered plasma concentrations of vitamin C. For example, Ballmer and colleagues26 reported the depletion of plasma vitamin C, but not of vitamin E, in response to cardiac operations. This contrasts with our study, in which the control patients had lowered vitamin C and lowered vitamin E concentrations, a result which supports those of other groups.19 27 28 The infusion of vitamin E did not prevent a decrease in vitamin C to subnormal concentrations. Some of this decrease may be attributed to haemodilution and some to the insufficiency of vitamin E to prevent depletion of the water-soluble part of antioxidative capacity in patients after coronary bypass operations. This raises the question of whether giving vitamin E without giving vitamin C at the same time is sufficient to block the formation of ROS. Indeed, combined dosage with vitamins E and C prevents lipid peroxidation after cardiac surgery.19 However, we wished to assess the effect of the exclusive use of vitamin E.

We studied patients with a high-risk profile. We felt that a difference of 20% in markers of myocardial injury would be meaningful, and a precise estimate of the sample size necessary to investigate this difference in markers of injury was used. The only finding was a 55% increase in MDA concentration 30 min after opening the aortic cross-clamp in both groups. If detection of the small differences in the other injury variables with sufficient power were desired, 350–3200 individuals would need to be studied. The same is true for finding significant differences in outcome variables such as non-Q-wave infarction. The only way to resolve this problem would be to conduct a new randomized trial with large numbers of patients. We doubt that the expected findings would justify such a study.

Conclusion
Our data show that parenteral administration of vitamin E beginning 12–16 h before surgery and extending 48 h into the postoperative period prevents postoperative depletion of vitamin E but not of vitamin C, and that the substitution of vitamin E at a normal serum concentration does not improve biochemical markers or clinical outcome in patients after cardiac surgery.


    Acknowledgement
 
This study was supported in part by funding from Fresenius Kabi Austria GmbH, Graz, Austria.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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