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Original Paper |
Department of Clinical Nutrition (C.C.T., J.S.H., M.A.M.) Rush Presbyterian St. Lukes Medical Center, Chicago, Illinois
Department of Cardiovascular Thoracic Surgery (W.P., Jr) Rush Presbyterian St. Lukes Medical Center, Chicago, Illinois
Address reprint requests to: Christine C. Tangney, PhD, Department of Clinical Nutrition, Rush Presbyterian St. Lukes Medical Center, 1743 West Harrison Street, Chicago, IL 60612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Methods: A consecutive sample of 38 patients undergoing CABG surgery at a Midwest tertiary care hospital was enrolled. Patients receiving blood transfusions before or during surgery were excluded.
Results: Plasma vitamin E/total lipid ratios rose with reperfusion, remained elevated immediately following bypass, and fell to preoperative concentrations by 24 hours. Plasma vitamin E/total cholesterol levels varied little throughout this time course. Both plasma uric acid and ascorbate concentrations (corrected for hemodilution) also rose by the preischemic interval, and remained elevated until a return to preoperative levels by 24 hours. Corrected malondialdehyde (MDA) concentrations rose by pre-ischemia but returned more quickly to preoperative levels. Atrial appendage tissue vitamin E concentrations bore a significant relationship to those of plasma prior to surgery (r=+0.49, p=0.004). Reported supplement use, plasma concentrations and body mass index contributed to the variability in atrial tissue concentrations of vitamin E.
Conclusions: In short, when not confounded by transfusions or hemodilution, several peripheral indices of antioxidants increase with the reperfusion segment of CABG procedure and return to baseline levels within 24 hours of surgery. Parallel changes in MDA were observed. The observed changes are consistent with the hypothesis that oxidative stress accompanies the ischemia-reperfusion components of the CABG procedure.
Key words: coronary artery bypass grafting, aortocoronary bypass, cardiopulmonary bypass, vitamin E, alpha-tocopherol, ascorbic acid, malondialdehyde
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Several clinical studies have examined the potential benefits of antioxidant supplementation in the setting of CABG because several markers of free radical production, such as H2O2 concentrations and malondialdehyde (MDA) concentrations, reportedly rose during surgery, lending support to the hypothesis of oxidative stress with ischemia-reperfusion [10–14]. Preoperative ingestion of vitamin E and C supplements attenuated the observed increases in such markers following ischemia [10,11,13]. Functional outcomes more directly related to the noted myocardial injury following CABG, specifically, fewer perioperative infarctions and less creatine kinase MB release, have also been reported for patients who consumed vitamin C and E supplements prior to surgery when compared to patients without such pretreatments [13]. In addition, two groups have demonstrated that myocardial tissue (atrial appendage) vitamin E concentrations are reduced with ischemia and/or reperfusion during CABG [15–17].
The present study was designed to assess prospectively whether the CABG procedure alters plasma vitamins E and C concentrations of adult patients through determinations at time points before, during and following CABG. Vitamin concentrations were determined at four time points during reperfusion because free radical formation is thought to occur at that time [18]. Unlike several previous reports, plasma measures were corrected for the hemodilution that accompanies CABG, and patients receiving blood transfusions just prior to or during surgery were excluded. Finally, we report the vitamin E content of myocardial tissues obtained at the onset of surgery for comparison with preoperative plasma vitamin E and C concentrations in order to assess whether plasma concentrations reflect myocardial tissue content.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A brief questionnaire was administered to each patient within 20 days of their operation regarding usual multivitamin or individual supplement use, smoking status (current, former or non-smoker) and alcohol consumption (amount per day or week). A current smoker was defined as one who has smoked cigarettes within the past year; a former smoker as one who quit more than a year ago; and a nonsmoker as one who had never smoked or quit more than 10 years ago. Current alcohol consumption was coded as number of drinks per week or month. Less than 1 drink per month was coded as "0", 1 to 3 drinks per week as "1", 4 to 20 drinks per week or more than 3 drinks per week but less than 3 drinks per day as "2" and more than 3 drinks per day as "3". The categories are similar to those reported by Fuchs and colleagues [19].
CABG Procedures
All CABG procedures were performed using cardiopulmonary bypass (CPB). The induction of general anesthesia and the coronary bypass procedure were carried out as described routinely [20]. The right atrial appendage was excised for sampling prior to placement of the venous cannula. Moderate hemodilution (hematocrit 22 to 24%) and moderate systemic hypothermia (core temperature 28 to 32°C) were used during CPB. Cardiac arrest was achieved by delivering a high-potassium-containing blood cardioplegic solution antegrade into the aortic root at a temperature of 5.9±0.5°C. The duration of aortic cross-clamping (termed the ischemic period) was recorded for each procedure.
Blood Sampling and Preparation
Blood specimens were obtained from arterial catheters at the following times: 1) pre-bypass or preoperative period (before sternotomy and heparin administration); 2) pre-cross clamp or pre-ischemia (5 minutes prior to cross-clamp); 3-6) 2, 5, 10, 20 minutes after cross-clamp release (reperfusion period); 7) immediately post-CPB (following chest closure and following protamine infusion); 8) 24 hours postoperatively (Table 1). Samples were collected by the perfusionist at the prescribed time intervals into vacutainers containing EDTA. Tubes were placed on ice and protected from daylight.
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For vitamin C analyses, plasma aliquots were immediately treated with 6% (w/v) metaphosphoric acid; tubes were then centrifuged at 3000xg for 6 minutes to precipitate protein. Timely centrifugation of blood and metaphosphoric acid treatment of plasma were necessary precautions because of the noted lability of vitamin C [23]. Deproteinized plasma aliquots are quite stable when stored at -70°C for several months [24,25]; in our laboratory, analyses of deproteinized control plasma pools after 2 months showed no significant changes in ascorbic acid content.
Quantification of serum creatine phosphokinase (CK) concentrations were performed routinely 24 hours postoperatively [T8] as well as 48 and 72 hours following CABG. CK elevations were considered clinically significant for possible myocardial injury if the CK-MB isozyme concentration was equal to or greater than 50 U/L [26].
Myocardial Tissue Biopsies and Preparation
The right atrial appendage biopsy sample consisted of tissue excised beyond the suture used for cannula insertion during cannulation for CPB. Specimens were obtained immediately before cross-clamping (or the ischemic period) and placed in saline on ice and stored away from light. Upon receipt to the laboratory, tissues were rinsed and frozen in liquid nitrogen and stored at -70°C until analyses were performed. Analytes stored in this manner are stable for several months [27,28].
Atrial tissue samples were thawed, weighed, minced finely and homogenized in a small volume of 1% (w/v) aqueous EDTA. Aliquots were saponified and extracted with hexane for chromatographic determination of vitamin E, specifically alpha- and gamma-tocopherols (high-performance liquid chromatography or HPLC) and fatty acids (gas-liquid chromatography). A second and third aliquot of the tissue homogenate were removed for standard protein determinations as described earlier [28].
Total Ascorbic Acid (Vitamin C), Vitamin E, and Lipid Analyses
Ascorbic acid concentrations were measured in deproteinized plasma by the 2,4-dinitrophenylhydrazine method first reported by Roe and Kuether [29] and modified by Gunther et al [30]. No ascorbic acid determinations were performed on atrial tissue specimens.
Alpha- and gamma-tocopherol concentrations in plasma and atrial tissue were quantified by a HPLC assay using fluorescent detection [28,31]. The aqueous phase of atrial tissue extracts were further acidified, extracted, and derivatized for fatty acid analyses by gas-liquid chromatography as described earlier [28].
Plasma total lipid values were calculated as twice the sum of total cholesterol (TC) and triacylglycerol (TG) concentrations as reported by Thurnham et al [32]. Plasma TC [33] and TG [34] concentrations were determined by standardized enzymatic methods routinely used in our laboratory.
Plasma Malondialdehyde (MDA) and Uric Acid Concentrations
Thawed plasma aliquots (0.25ml) were used to determine MDA concentrations according to modifications of the method of Tatum and co-workers [35]. Plasma samples were treated with 2-thiobarbituric acid (TBA) to yield a TBA-complex that was extracted with isobutanol and mixed with methanol (2/1, v/v) prior to injection into the HPLC system. The HPLC system consisted of a C18 reversed-phase column, a Waters 740 (Millipore, MA) fluorescence detector set at 515 nm for excitation and 550 nm for emission and the mobile phase of a 1/1 methanol/water (v/v) mixture containing tetrabutylammonium dihydrogen phosphate (0.05%, w/v).
Uric acid concentrations were determined by the Sigma (Procedure 685, Sigma Chemical Co., St. Louis, MO) enzymatic procedure using uricase and peroxidase. The normal ranges for plasma uric acid concentrations for men are 214 to 458 umol/L and 149 to 405 umol/L for women [36].
Data Analyses
Plasma vitamin C measurements are reported as corrected values (CVC) to account for the hemodilution secondary to the crystalloid pump-priming solution used for the extracorporeal CPB circuit. Crude or uncorrected (UVC) values were multiplied by the ratio of the initial hematocrit to the hematocrit at the specified time. MDA and uric acid concentrations were corrected in the same manner. Albumin was not used to correct for hemodilution because the extracorporeal unit is primed with albumin and patients may receive albumin transfusions during surgery or post-operatively. Plasma total vitamin E or TVE concentrations (sum of gamma- and alpha-tocopherols) were expressed in several ways: as uncorrected or crude values, as a ratio of TVE to total lipids [32] and, for comparison with other researchers, as a ratio of TVE to TC [12,14].
Statistical Analyses
Descriptive statistics and all subsequent analyses were performed with the Statistical Package for Social Sciences, SPSS-PC, version 5.01 (SPSS Inc., Chicago, IL). When histograms of variables were examined for normality, all variables were log (log10) transformed, except plasma vitamin C and uric acid. Comparisons of preoperative (T1) concentrations of CVC, TVE, TC and TG according to gender, race, smoking status, supplement use, and alcohol use were performed using either Students t test or one way ANOVA tests. Similar tests were used to contrast total CK or CK-MB values of patients at T8 or 24 hours postoperatively.
To examine the effects of CPB and reperfusion on circulating antioxidants and peroxidation indices, plasma measures were compared across time [including T1 through T8] by repeated-measures ANOVA (rMANOVA). Because no significant differences were observed among the four reperfusion timepoints for any of the plasma measures (except uncorrected TG), the average of values at all four reperfusion intervals [T3-T6] were used for subsequent analyses. Bonferonni-adjusted paired t tests using the listwise missing cases option were performed to identify which times were different when an overall significant within-subject time effect was observed with rMANOVA. The listwise option was selected to ensure that an equal number of observations for the a priori identified time points were compared (
=0.012 for all but TG with an =0.006).
Atrial tissue vitamin E indices were examined in relation to reported supplement use, smoking status and gender with one-way ANOVA. Relationships between preoperative plasma values and atrial vitamin E concentrations were examined with Pearson’s product-moment correlation tests. Stepwise multivariate linear regression analyses were used to identify which measurements might best predict the magnitude of change in lipid peroxidation indices, in particular, plasma MDA concentrations during the CABG procedure. Uric acid concentrations may represent an antioxidant since urate or the salt of uric acid is a good peroxy radical scavenger [37]. The magnitude of change was defined as either the peak or nadir value or percent change from preoperative values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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0.8 and TVE, 0.5 mg/dL or 11.6 umol/L) [38]. The TVE/lipid ratio may be more appropriate in the clinical setting in which several patients were frankly hyperlipidemic (Table 3) [32]. With respect to preoperative vitamin C concentrations, 12% of patients exhibited concentrations suggestive of marginal vitamin C status (
0.4 mg/dL or 23 umol/L) [38].
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Comparisons of Plasma Lipids and Vitamins as a Function of CPB Period
In Table 3, both uncorrected and corrected values for selected analytes are included to demonstrate the impact of hemodilution. In nearly every patient, preoperative [T1] levels of lipids and vitamins were significantly greater than those observed at any time throughout CPB or postoperatively [T2–T8]. With dilution correction, TVE/TC values vary little throughout the CPB procedure, which may reflect the similar declines in both numerator and denominator—a 43% decrement in TC, and 38% for TVE. On the other hand, while there was no difference in TVE/lipid values between T1 and T2, the ratio rose significantly during reperfusion [average of T3-T6], p=0.002, and remained higher immediately after bypass [T7], p=0.008. By 24 hours after the CABG [T8], values for TVE/lipid ratios were no different from those prior to surgery. Corrected vitamin C (CVC) concentrations exhibited a slightly different pattern with a significant rise from baseline to T2, and lesser, yet still significant elevations throughout the next two time intervals [average T3-T6 and T7] until concentrations returned to baseline 24 hours later at T8. Similar to the CVC pattern, MDA concentrations rose markedly by T2 and were significantly higher throughout reperfusion timepoints when compared to those at T1, but the T7 and T8 values were no different from those observed at T1. Corrected uric acid concentrations reflect a pattern more akin to that observed for CVC with a significant rise at T2 that was maintained to the conclusion of surgery, but returned to preoperative value after 24 hours.
CK concentrations of patients averaged 561.6±775.3 U/L (n=32) 24 hours after surgery; only two patients had CK-MB values that exceeded 50 U/L. There were no significant correlations observed between any of these markers of myocardial injury and preoperative antioxidant measures (data not shown). Nor were there any significant differences in CK or CK-MB fractions at T8 according to gender, supplement use, smoking or alcohol status (data not shown).
Subject Comparisons of Atrial Vitamin E Measures
Reported supplement use clearly discriminated between atrial tissue vitamin E concentrations whether expressed in terms of wet weight or protein (Table 5). As expected, alpha-tocopherol represented the major vitamin E compound observed in this tissue. Smoking status, gender or current alcohol use did not appear to influence tissue vitamin E concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The main outcomes of this investigation include increases in corrected plasma vitamin E and C concentrations during reperfusion and a return to baseline values within 24 hours of surgery. Similar changes in corrected MDA and uric acid concentrations were also observed. Unlike several other reports [14,40], the longitudinal changes in plasma measures are presented only for patients who did not receive blood transfusions. Atrial alpha-tocopherol or TVE concentrations were highly related to those found in plasma preoperatively [T1].
Uncorrected vitamin E values were markedly lower than those before surgery [T1] at all time points throughout surgery including 24 hours postoperatively [T8]. This finding confirms that of others [10,13,40]. Only one other group [40] reported lipid-adjusted TVE levels and found little change in this index across the course of surgery and 24 hours postoperatively [T8]. In the present report, TVE/lipid ratios rose with reperfusion to a maximum at T7, followed by a fall to preoperative levels by 24 hours [T8]. Two groups also expressed concentrations in relation to total cholesterol (TVE/TC), as we did. We found little change in TVE/TC ratios across time in the present study in agreement with one of these reports [12]. A different pattern was reported by Murphy and co-workers [14], who found significant elevations in alpha-tocopheryl quinone (an oxidized metabolite of alpha-tocopherol) and in alpha-tocopheryl quinone/alpha-tocopherol ratios during CPB with an accompanying increase in MDA concentrations at reperfusion. These findings are difficult to evaluate because all subjects received blood transfusions intra-operatively, so that the changes in plasma concentrations might be obscured by such treatments.
Only one other report [10] supports our finding that CVC concentrations rise from those prior to surgery, remain elevated throughout CPB, and return to preoperative levels by 24 hours post surgery. However, the confirmation is less reassuring because we question whether hemodilution corrections were made in that report [10]. We were able to describe a peak CVC concentration at the pre-ischemic interval [T2], followed by a gradual decline to preoperative concentrations at 24 hours [T8]. This pattern varies from that reported by Ballmer’s group who also reported CVC concentrations: they describe a gradual decline in CVC during the CABG with a nadir 24 hours postoperatively [40]. In the present study, we observed a nadir at 24 hours [T8], but only with UVC; CVC values at T8 reflect a return to preoperative concentrations. The present researchers cannot suggest a reason for the different CVC patterns other than the inclusion of patients who had received blood transfusions in the report by Ballmer and associates [40]. In agreement with others [10,13,14], we suggest that vitamin C concentrations increase early during surgery to repair/reduce oxidized vitamin E. The observed decline following the pre-ischemic period (although CVC were still higher than preoperative concentrations) probably reflects the gradual removal of oxidant stress with the ensuing reperfusion period.
Vitamin C has been shown to regenerate vitamin E from its radicals [10,41,42]. Similar actions have been noted for uric acid, another radical scavenger [37]. Furthermore, these interactions may explain the observed inverse association between percent change in uric acid concentrations with TVE/lipid or alpha tocopherol/lipid ratios; that is, those patients with the highest preoperative vitamin E/lipid ratios experienced the smallest changes in uric acid concentrations throughout surgery.
To the best of our knowledge, only three groups [11,13,40] report plasma uric acid concentrations of patients during cardiac surgery. In one of these studies, hemodilution corrections were clearly used for uric acid [11]; in the second [13], it is not clear whether any corrections were made. In the third [40], uric acid levels were not corrected, but those of vitamin C were corrected. Our uncorrected urate levels (data not shown) decline throughout surgery and immediately following surgery [T2-T7], but rise and return to levels observed prior to surgery by 24 hours, similar to the pattern reported by Ballmer and colleagues [40]. The pattern for corrected urate levels resembles those reported by Barta’s group [11] in that levels rose during surgery, even though different timepoints were selected.
As a reflection of oxidative stress during the pre-ischemic and reperfusion intervals, significant increases in corrected MDA concentrations from baseline were observed in the present study. The dramatic rise in MDA concentrations during reperfusion is consistent with that observed by Coghan’s group [12], who measured thiobarbituric acid-reacting substances (TBARS), a less specific assay than the one used in the present study.
Atrial tissue TVE concentrations were highly variable among subjects whether expressed in terms of wet weight, protein or lipid. Such variability has also been noted by Barsacchi and coworkers [15]; these investigators state that the variability in basal tissue concentrations (expressed per mg dry weight, 355±249 pmol vitamin E) appeared independent of age, sex and clinical status. On the contrary, we found that some of the variability in atrial vitamin concentrations is attributable to anthropometric (BMI) and clinical (number of grafts or NYHA classification) status. Any anthropometric indicator that reflects the degree of adiposity can be an important determinant of the relationship between plasma and tissue tocopherol content [43,44], in addition to plasma lipids. Plasma tocopherol measures incorporating a correction for plasma lipids has been long recognized as the preferred expression [45] because tocopherol transport to peripheral tissues is mediated by lipoproteins, primarily through two mechanisms—a lipoprotein lipase and a LDL-receptor pathway [46]. Thus, plasma vitamin E measures corrected for total lipids serve as appropriate markers of available vitamin E for myocardial tissue uptake, as it did in our regression model.
Barsacchi’s group [15] described a decline in atrial tissue content with ischemia and reperfusion because two specimens were obtained from each patient. Tissue vitamin E concentrations declined with ischemia and appeared to rise to slightly higher concentrations with reperfusion. In another series of studies by Mickle’s group [16,17], examination of multiple ventricular myocardial specimens demonstrated a small but significant decline in tissue alpha-tocopherol concentrations with the onset of reperfusion that returned to preoperative concentrations by 20 minutes of reperfusion. The decline in tissue alpha-tocopherol in early reperfusion and its "normalization" by 20 minutes of reperfusion corresponds with the pattern of myocardial gain and loss of alpha-tocopherol derived from arterial-coronary sinus differences described by Coghlan’s group [12]. The gradual increase in arterial TVE/lipid ratios during reperfusion [T3–T6] observed in our study supports this pattern.
In summary, the biochemical changes accompanying CABG surgery observed in this report are in accordance with the free-radical theory. Unlike many earlier reports, these data are not confounded by blood transfusions or hemodilution. Although not measured at multiple time points throughout CABG surgery, myocardial alpha-tocopherol concentrations bore a significant relationship with preoperative plasma concentrations. Unlike a recent report [47], smoking status did not discriminate between tissue vitamin E concentrations or plasma vitamin C or plasma MDA concentrations. This discrepancy may be due to the smaller number of current smokers in our study sample. Supplement use also appeared to explain much of the variability in myocardial tissue concentrations. Based upon the work of Mickle and colleagues [16], a minimum of 14 days of preoperative vitamin E supplementation is required to effect substantial increases in myocardial tissue vitamin E content. The relationship between supplement use and both myocardial tissue and preoperative plasma concentrations noted in Tables 4 and 5 lends support to the potential for vitamin deposition into these tissues. Further interventions using vitamin E supplements for a minimum of 14 days prior to scheduled CAB as well as follow-up of such patients with respect to ischemic events are necessary to evaluate the clinical significance of antioxidant treatment in myocardial injury following CABG.
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ACKNOWLEDGMENTS |
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Received March 1, 1997. Accepted August 1, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol.
Cardiovasc Res
25:
89–92,
1991.-tocopherol supplements decrease plasma gamma-tocopherol levels in humans.
J Nutr
115:
807–813,
1985.
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