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Anesth Analg 2002;94:89-93
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Tissue Antioxidant Capacity During Anesthesia: Propofol Enhances In Vivo Red Cell and Tissue Antioxidant Capacity in a Rat Model

Tim D. Runzer, MD*, David M. Ansley, MD*, David V. Godin, PhD{dagger}, and Gordon K. Chambers, MD{ddagger}

Departments of *Anesthesia and {dagger}Pharmacology and Therapeutics, University of British Columbia; and Department of {ddagger}Healthcare and Epidemiology, Center for Clinical Epidemiology and Evaluation, Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, Canada

Address correspondence and reprint requests to David M. Ansley, MD, University of British Columbia Department of Anesthesia, Room 3200, 3rd floor, 910 West 10th Ave., Vancouver, BC, Canada V5Z 4E3. Address e-mail to daansley{at}interchange.ubc.ca


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The effects of anesthesia on ischemia-reperfusion injury are of considerable scientific and clinical interest. We examined the effects of propofol (known to possess antioxidant activity) and halothane (devoid of antioxidant activity in vitro) on tissue and red blood cell (RBC) antioxidant capacity. Adult male Wistar rats were anesthetized with halothane 0.5%–1.0% (n = 7), propofol 500 µg · kg-1 · min-1 with halothane 0.25%–0.5% (small-dose propofol; n = 9), or propofol 2000 µg · kg-1 · min-1 (large-dose propofol; n = 8) for 45 min. Blood and tissue samples of liver, kidney, heart, and lung were then harvested for in vitro exposure to a peroxidizing agent. Red cell malondialdehyde and tissue thiobarbituric acid reactive substances were determined spectrophotometrically. Antioxidant capacities of blood and tissues in the Large-Dose Propofol group, and of blood and all tissues except lung in the Small-Dose Propofol group, were increased significantly compared with halothane (P < 0.003). The increases in tissue antioxidant capacities varied in their magnitude: RBC > liver > kidney > heart > lung. There was a high correlation between changes in RBC susceptibility to oxidative damage and corresponding changes in tissues. These findings demonstrate that large-dose propofol significantly enhances tissue antioxidant capacity, and RBC antioxidant capacity can serve as a functional measure of tissue activity, in vivo.

IMPLICATIONS: We designed this study to investigate the antioxidant effects of propofol in various tissues in a rat model. Pretreatment of animals with propofol led to a reduction in the susceptibility to an in vitro oxidative stress of five different tissues investigated, demonstrating the drug’s ability to limit oxidative injury. This may have future application in limiting organ dysfunction after periods of tissue ischemia (which results in oxidative damage).


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
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Organ dysfunction after ischemia-reperfusion (IR) may result from oxidant-mediated cellular damage (1,2). Recent interest has been focused on the use of antioxidants to prevent such injury. Propofol, a highly lipid soluble anesthetic, possesses antioxidant activity in vitro and in vivo (3–5). This property makes it suitable to test for efficacy in clinical and experimental settings of IR injury (6–8).

Previous studies in our laboratory have demonstrated that propofol can increase the antioxidant capacity of red blood cells (RBCs) of both swine and humans in vivo (3). We have also shown this effect under clinical conditions of cardiopulmonary bypass (4). Other investigators, using cultured rat tissues, have found that propofol similarly protects heart, liver, kidney, lung, and brain against forced peroxidation (6). It is not clear from these previous studies, however, whether the protective effect of propofol on RBCs is indicative of (and could be used as a measure of) a generalized antioxidant effect at the level of tissues in vivo. Clearly, this would be a required property of any antioxidant used to modify IR injury in a clinical setting.

The purposes of the current study were: 1) to determine the dose response of propofol on antioxidant status in vivo, and 2) to determine the relationship between alterations in tissue and RBC antioxidant capacity in vitro.


   Methods
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 Methods
Results
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This study was approved by the Institutional Committee on Animal Care at the University of British Columbia. Animals were housed 2–4 per cage in our animal care facility for at least 1 wk before study, under 12-h cycles of light and dark. They were allowed free access to water 24 h per day.

Male Wistar rats (250–300 g, n = 24) were anesthetized in an anesthetic bell jar by using 2% halothane in oxygen. After anesthetic induction, the trachea was exposed by cutdown and intubated with a 2.5-cm, 14-gauge IV catheter. All animals were ventilated by using a Harvard Apparatus Rodent Ventilator (Holliston, MA)(7 mL/kg tidal volume at 60 breaths/min; fraction of inspired oxygen = 1.0). The left carotid artery and right internal jugular vein were surgically exposed and cannulated for pressure measurement and IV infusion, respectively. The arterial pressure and heart rate were recorded continuously on a polygraph (Grass Instruments, West Warwick, RI). After 45 min of equilibration under halothane anesthesia, the animals were allocated into one of three anesthetic groups: Group 1 (n = 7), 0.5%–1% halothane; Group 2 (n = 9), small-dose propofol with halothane (500 µg · kg-1 · min-1 and 0.25%–0.5%, respectively); or Group 3 (n = 8), large-dose propofol (2000 µg · kg-1 · min-1) without halothane. The total volume of infused fluid was 4 mL/h, consisting of normal saline with or without propofol depending on the anesthetic group. After 45 min of anesthesia, blood samples were obtained by cardiac puncture, using heparin as anticoagulant. The lower lobe of the right lung, the heart, the right hepatic lobe, and the left kidney were immediately harvested and placed in ice-cold 50 mM Tris-0.1 mM EDTA buffer, pH 7.4. RBC antioxidant capacity was determined by exposure of RBC membrane lipids to the peroxidizing agent t-butylhydroperoxide (t-BHP). This results predominantly in malondialdehyde (MDA) formation. The concentration of MDA can be determined by a measurement of optical densities with a mathematical correction for interfering substances (9). Oxidation of other tissues by t-BHP results in the formation of numerous lipid byproducts, simply termed thiobarbituric acid reactive substances (TBARS). The concentration of TBARS in the sample is estimated from the absorbance at 532 nm (5).

Tissue samples (400 mg) from each organ were homogenized on ice in 4 mL of the Tris-EDTA buffer by using a Polytron (PT-10, Brinkman Instruments, Canada) homogenizer for 30 s at 25% power. The resulting homogenates were used for in vitro forced peroxidation and subsequent determination of TBARS as described previously (10). In brief, 400 µL of tissue homogenate were combined with 400 µL of t-BHP (in 0.9% saline/2 mM sodium azide) to produce t-BHP concentrations ranging from 0.125 to 10 mM. These suspensions were incubated for 30 min at 37°C, then 400 µL of cold 28% (w/v) TCA-0.1 M sodium arsenite was added. The mixture was centrifuged at 12,000g for 5 min at 4°C, then 800 µL of supernatant was removed and added to 400 µL of thiobarbituric acid (0.5% in 25 mM NaOH). The samples were boiled for 15 min, and the absorbance at 532 nm was measured spectrophotometrically.

Blood samples were centrifuged at 1000g for 5 min at 4°C, plasma and white cells removed, and red cells washed 3 times with 0.9% saline-2 mM sodium azide. Aliquots of packed red cells (40 µL) were weighed, resuspended in 360 µL of saline-azide, and preincubated for 5 min at 37°C. The reaction was initiated by the addition of 400 µL of t-BHP (prepared in saline-azide solution) to produce t-BHP concentrations in the range of 0.125 to 2.5 mM. Samples were incubated for 30 min at 37°C, then the reaction was terminated by the addition of 400 µL of 28% TCA-0.1 M sodium arsenite. After centrifuging at 12,000g for 5 min at 4°C, 800 µL of supernatant was removed and combined with 400 µL of thiobarbituric acid solution (0.5% in 25 mM sodium hydroxide). The samples were boiled for 15 min and MDA levels determined spectrophotometrically by measurement of optical densities at 532 and 453 nm as previously described (8).

In a separate pilot experiment, blood obtained by cardiac puncture from a halothane-anesthetized rat was centrifuged and washed as described above. Packed red cells (300 µL) were resuspended in 300 µL of propofol (diluted with isotonic saline) to obtain final concentrations of propofol ranging from 50 to 400 µM. These solutions were incubated at 37°C for 15 min, followed by centrifugation at 12,000g for 5 min. The red cell pellets were then washed and subjected to the forced peroxidation protocol as described above.

All analyses were conducted by using the Statistical Analysis System(SAS, Cary, NC). Relationships among the three groups of Small-Dose Propofol, Large-Dose Propofol, and Halothane looking at RBC and tissue antioxidant capacity levels were analyzed by using Pearson correlation coefficients. The data were analyzed for intergroup differences in red cell MDA level, and TBARS formation in tissues. Analysis of variance was used to determine significant differences among groups and Student-Newman-Keuls multiple range test was used to identify specific differences. All analyses were undertaken using an {alpha} of 0.05 level of significance.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
All animals were hemodynamically stable throughout the experiment. None of the animals required vasopressor support.

Forced peroxidation of red cells and tissues with increasing concentrations of t-BHP yielded increasing levels of MDA and TBARS, respectively (Figs. 1,2). The dose-response curves have a sigmoid shape. If larger concentrations of t-BHP are used, MDA and TBARS begin to decrease (data not shown). This may be attributable, at least in part, to decreased absorbances at large t-BHP concentrations because of the bleaching effect of peroxides. A concentration of t-BHP of 1.5 mM yields MDA and TBARS within the steep portions of these curves. This concentration of peroxidizing agent, therefore, is sufficient to produce a response in all tissues while minimizing the effect of sample bleaching that occurs at large peroxide concentrations, and was used to compare the different anesthetic regimes.



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  		Figure 1. Malondialdehyde (MDA) formation in red blood cells (RBC) as a function of t-butylhydroperoxide (t-BHP) concentration for each anesthetic group. Data were expressed as means ± SD. Low-Prop = low-dose propofol, Hi-Prop = high-dose propofol.

 


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  		Figure 2. Thiobarbituric acid reactive substances formation (as reflected by absorbance at 532 nm) as a function of t-BHP concentration for tissues in each anesthetic group. Data were expressed as means ± SD.

 
MDA production by red cells after a challenge with 1.5 mM t-BHP decreased as the propofol dose increased (Fig. 3). The Large-Dose Propofol group shows a greater than threefold decrease in MDA production relative to the Halothane group.



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  		Figure 3. Malondialdehyde (MDA) formation in rat red blood cells (RBCs) at 1.5 mM t-butylhydroperoxide according to anesthetic group. Error bars indicate SD, *P = 0.003 high-dose propofol (Hi-Prop) versus halothane; Student-Newman-Keuls multiple range test. Lo-Prop = low-dose propofol.

 
Small-dose propofol (500 µg · kg-1 · mL-1) significantly decreased t-BHP-induced TBARS formation in all tissues except lung (P < 0.003). A similar, but even greater, significant decrease (P < 0.003) in TBARS formation was observed in all tissues with large-dose propofol (2000 µg · kg-1 · min-1) (Fig. 4). Different tissues demonstrated quantitatively different responses to the same dose of propofol (relative to halothane) (Fig. 5). Comparing tissues from the Large-Dose Propofol group to those from the Halothane group, the relative protection in liver (63%) far exceeded that in lung (26%), with kidney and heart showing intermediate responses to propofol (54% and 35%, respectively). The relative reduction in TBARS for liver, kidney, and heart exposed to small-dose propofol versus halothane demonstrated the same order of tissue sensitivities. The relative protection, however, was less in the Small-Dose Propofol group (30% for liver, 21% for kidney, and 13% for heart).



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  		Figure 4. Formation of thiobarbituric acid reactive substances in various tissues (represented as absorbance at 532 nm) at 1.5 mM t-butylhydroperoxide with groupings according to anesthetic type. Error bars indicate SD, *P = 0.003 propofol versus halothane; Student-Newman-Keuls multiple range test.

 


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  		Figure 5. Percent reduction in thiobarbituric acid reactive substances (TBARS) between halothane and large-dose propofol and halothane and small-dose propofol.

 
In a preliminary pilot experiment (data not shown), we demonstrated that in vitro peroxidation of red cells is inhibited by propofol in a dose-dependent manner in the concentration range of 50 to 400 mM. However, red cell MDA formation could not be fully inhibited by propofol.

Although intralipid, the vehicle for propofol, was not specifically evaluated as an antioxidant in this study, previous investigations, including our own, have demonstrated that it does not possess such activity (3,11).

Using Pearson product moment correlations, we found strong positive linear correlations between RBC and tissue antioxidant capacity for each tissue studied within each anesthetic group (Table 1). The Pearson coefficients ranged from 0.95771 to 0.99691.


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  		Table 1. Pearson Correlation Coefficients for RBCs and Individual Tissues in Each Anesthetic Group
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of this study show that propofol is capable of increasing the antioxidant capacity of RBCs and tissues in an in vivo rat model. The antioxidant effect may be attributable to the ability of propofol to capture electrons from free radicals and become a relatively stable intermediate by virtue of its phenolic structure (12). Previous studies support the claim that propofol acts primarily as a free radical scavenger as opposed to a modulator of enzymatic antioxidant systems (12).

The antioxidant capacities of the nonhematologic tissues were variably affected by propofol. Our study has demonstrated the relative response of tissues to the protective effects of propofol to be: liver > kidney > heart > lung. This finding may be explained by differences in the inherent susceptibility of each tissue to lipid peroxidation. De La Cruz et al. (6) have made a similar observation in an in vitro study of the effects of propofol on the forced peroxidation of various rat tissues. However, they found the relative response of tissues to be: heart > lung > kidney > liver. This discrepancy may be explained by differences in regional blood flow to individual organs during the in vivo phase of our experiment, which may affect the amount of drug accumulating in the tissues. These findings imply that tissues may show variable functional protection after propofol exposure. Clearly, however, the effects of propofol on organ functional preservation, after an ischemia reperfusion challenge, remain to be elucidated.

The red cell data are in keeping with previous studies in our laboratory, which demonstrated similar antioxidant enhancement of red cells from both swine and humans (3,8). The fact that an antioxidant effect of propofol was demonstrable on red cells incubated with anesthetic in vitro suggests that the antioxidant action of the anesthetic in vivo is direct, so that postulation of the involvement of secondary mediators of the effect seems unnecessary. In the present study, blood propofol concentrations were not determined. However, the dose dependence of the antioxidant effect of propofol on red cells allows an estimation of the levels of anesthetic attained in the blood. The maximal degree of reduction in red cell MDA production, when comparing the Halothane and Large-Dose Propofol groups, was 68% in this study. In our pilot investigation of in vitro forced peroxidation of rat RBCs, this same degree of protection was observed with blood propofol concentrations between 200 and 300 µM. In this same pilot study, the reduction in MDA formation in red cell membranes seemed to reach a maximum at approximately 80% with a propofol concentration of 400 µM.

There was a close correlation between red cell and tissue antioxidant capacity during anesthesia. This correlation could be of practical significance because it implies that the level of tissue protection provided by a given dose regimen of propofol can be reliably predicted by an assay of RBC antioxidant capacity. This may prove to be of value in future investigations of the effects of propofol on the preservation of organ function. Clinically, given the minimally invasive nature of the procedure, the estimation of red cell antioxidant status would allow a means of optimizing and individualizing interventions using propofol (or other antioxidants) for the protection of tissues at risk of sustaining IR injury, for example, during coronary revascularization or organ transplantation.

In conclusion, this study demonstrates that propofol increases the antioxidant capacity of RBCs and, in parallel, that of liver, kidney, heart, and lung in rats. We have shown that individual tissues vary in their response to the protective effects of propofol against forced peroxidation. Finally, there is a strong correlation between the antioxidant capacity of RBCs and each of the four other tissues studied. Future experimental and clinical studies should be conducted assessing whether pharmacologic enhancement of antioxidant capacity with propofol confers organ protection after IR.


   Acknowledgments
 
This project was supported, in part, by the Glaxo-Wellcome Inc. Canadian Research Award in Anesthesia.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ross S, Foex P. Protective effects of anaesthetics in reversible and irreversible ischaemia-reperfusion injury. Br J Anaesth 1999; 82: 622–32.[Abstract/Free Full Text]
  2. Forman MB, Virmani R, Puett DW. Mechanisms and therapy of myocardial reperfusion injury. Circulation 1990; 81: IV69–78.
  3. Ansley DM, Lee J-U, Godin VD, et al. Propofol enhances red cell antioxidant capacity in swine and humans. Can J Anaesth 1998; 45: 233–9.[Web of Science][Medline]
  4. Murphy PG, Myers DS, Davies MJ, et al. The antioxidant potential of propofol (2,6-diisopropylphenol). Br J Anaesth 1992; 68: 613–8.[Abstract/Free Full Text]
  5. Green TR, Bennett SR, Nelson VM. Specificity and properties of propofol as an antioxidant free radical scavenger. Toxicol Appl Pharmacol 1994; 129: 163–9.[Web of Science][Medline]
  6. De La Cruz JP, Sedeno G, Carmona JA, Sanchez de la Cuesta F. The in vivo effects of propofol on tissular oxidative stress in the rat. Anesth Analg 1998; 87: 1141–6.[Abstract/Free Full Text]
  7. Bao YP, Williamson G, Tew D, et al. Antioxidant effects of propofol in human hepatic microsomes: concentration effects and clinical relevance. Br J Anaesth 1998; 81: 584–9.[Abstract/Free Full Text]
  8. Ansley DM, Sun J, Visser WA, et al. High dose propofol enhances red cell antioxidant capacity during CPB in humans. Can J Anaesth 1999; 46: 641–8.[Web of Science][Medline]
  9. Gilbert HS, Stump DD, Roth EF Jr. A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation. Anal Biochem 1984; 137: 282–6.[Web of Science][Medline]
  10. Godin VD, Garnett ME. Species-related variations in tissue antioxidant status. II. Differences in susceptibility to oxidative challenge. Comp Biochem Physiol 1992;103B:743–8.
  11. Mathy-Hartert M, Deby-Dupont G, Hans P, et al. Protective activity of propofol, Diprivan, and intralipid against active oxygen species. Mediators Inflamm 1998; 7: 327–33.[Web of Science][Medline]
  12. Mouithys-Mickalad A, Hans P, Deby-Dupont G, et al. Propofol reacts with peroxynitrite to form a phenoxyl radical: demonstration by electron spin resonance. Biochem Biophys Res Commun 1998; 249: 833–7.[Web of Science][Medline]
Accepted for publication August 28, 2001.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2002 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press