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Propofol and tourniquet induced ischaemia reperfusion injury in lower extremity operations

Turan, R.*; Yagmurdur, H.*; Kavutcu, M.; Dikmen, B.*

European Journal of Anaesthesiology: February 2007 - Volume 24 - Issue 2 - p 185–189
doi: 10.1017/S0265021506001347
Original Article
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Background and objective: Extremity surgery with tourniquet to provide a bloodless field may be a good human model for ischaemia reperfusion (IR) injury. The aim of this study was to investigate the effects of three different modes of propofol use on tourniquet induced IR injury in lower extremity operations.

Methods: Thirty-three consecutive ASA Grade I and II patients were randomized into three groups of 11 patients each. In the spinal group (Group S), after intrathecal anaesthesia, sedation was given with a propofol infusion at 2 mg kg−1 h−1 after a 0.2 mg kg−1 bolus dose and fentanyl 100 μg. In the general (Group G) and TIVA (Group T) groups, general anaesthesia was induced with propofol 2 mg kg−1 with fentanyl 100 μg and maintained with inhalation of halothane or infusion of propofol respectively. Venous blood samples were obtained at different time points for measurements of plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels.

Results: Plasma MDA levels were increased significantly in the Group G at 1 min before tourniquet release and 5 and 20 min after tourniquet release compared with before induction of general anaesthesia (baseline). Before intrathecal anaesthesia and before induction of general anaesthesia significantly decreased levels of MDA were observed both before and after tourniquet release compared to baseline. Plasma SOD and CAT concentrations were decreased significantly only at tourniquet release in the Group G compared with baseline. In the Groups S and T these enzymes were not changed significantly. Plasma GPx levels were not altered in any groups.

Conclusion: Propofol administration may inhibit lipid peroxidation and restore antioxidant enzyme levels in extremity surgery requiring tourniquet application.

*The Ministry of Health Ankara Research and Training Hospital, Clinic of Anaesthesiology and Reanimation, Ankara, Turkey

Gazi University School of Medicine, Department of Biochemistry, Ankara, Turkey

Correspondence to: Hatice Yagmurdur, Esat Cad. 102/10, Kucukesat, Cankaya, Ankara 06660, Turkey. E-mail: hyagmurdur@yahoo.com; Tel: +90 312 4473356; Fax: +90 312 4473356

Accepted for publication 31 July 2006

First published online 29 August 2006

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Introduction

Control of intraoperative bleeding with proximal tourniquet placement is a universal technique in extremity surgery. However, the release of a tourniquet causes an ischaemia reperfusion (IR) injury [1]. Postischaemic reperfusion injury is associated with the generation of reactive oxygen species (ROS) which damage cellular components and initiate the lipid peroxidation process [2]. Lipid peroxidation is the most important damaging effect of free radicals. Malondialdehyde (MDA) is an intermediate product of lipid peroxidation, and it can be used as a marker of free radical formation [3]. The tissue damage caused by the production of ROS can trigger several defence mechanisms. The first line defence mechanism includes antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [4]. These enzymes catalyse the conversion of ROS into less reactive species. Free-radical mediated tissue injury can be limited by the use of antioxidant therapy and several studies have suggested a positive role for antioxidant therapy in skeletal muscle reperfusion injury [5,6].

Propofol has potent antioxidant activity both in vitro and in vivo [7,8]. Propofol also attenuates tourniquet induced IR injury in human beings [9,10]. In all these previous studies propofol has been administered as an infusion and compared with other anaesthetic agents. We considered that propofol to be a promising agent against IR injury, and decided to investigate the efficacy of three different modes of propofol administration on tourniquet induced IR injury; as induction dose only, as part of a total intravenous (i.v.) technique and as low dose sedation.

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Methods

The study protocol was approved by the institutional review board of Ministry of Health Ankara Research and Training Hospital and all patients gave their verbal and written informed consent. Thirty-three consecutive ASA Grade I and II patients, aged between 18- and 60-yr old, undergoing unilateral lower extremity operation with use of a tourniquet were randomized into three groups of 11 patients each. The number of patients in each group were assessed by correlation regression analysis. None of the patients' histories indicated prior use of cigarette and antioxidants.

In the spinal group (Group S), intrathecal anaesthesia by 0.5% heavy bupivacaine 10–12.5 mg was performed first. When a sensory block level at T8–T10 was ensured by pinprick test, sedation was started with low dose propofol infusion 2 mg kg−1 h−1 after a 0.2 mg kg−1 bolus dose and fentanyl 100 μg. In the general (Group G) and TIVA (Group T) groups, general anaesthesia was induced with propofol 2 mg kg−1 and fentanyl 100 μg. Tracheal intubation was facilitated by vecuronium 0.1 mg kg−1 in both groups and the lungs were ventilated with 60% nitrous oxide in oxygen (10 mL kg−1 tidal volume, 10 breaths min−1, end-tidal CO2 34–37 mmHg). Halothane 0.6–1% was added to this gas mixture in Group G and an i.v. infusion of propofol at a rate of 10 mg kg−1 h−1, reducing to 8 and 6 mg kg−1 h−1 respectively at 10 min intervals was used in Group T. Mean arterial pressure (MAP) was maintained at 70–90 mmHg by infusing crystalloid solutions and peripheral oxygen saturation (SPO2) was above 96% throughout the operation in all patients. A tourniquet was applied at a pressure twice the systolic arterial blood pressure.

Blood samples were obtained from a peripheral vein in the upper extremities before spinal and induction of general anaesthesia (baseline), 1 min before tourniquet release (BTR) and 5 and 20 min after tourniquet release (ATR), for the measurements of MDA, SOD, CAT and GPx. Blood samples were centrifuged (1500 rpm) within 10 min and the supernatants were stored at −70°C until analysis within 2 weeks.

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Determination of lipid peroxidation

The thiobarbituric acid assay, which measures MDA, is the most commonly used method for estimating lipid peroxidation. MDA reacts with thiobarbituric acid to form a coloured complex. Thiobarbituric acid reactive substances were determined using the method described by Yoshioka and colleagues [11]. 0.5 mL plasma, 2.5 mL trichloroacetic acid (200g L−1) and 1 mL thiobarbituric acid (6.7g L−1) were mixed and boiled for 30 min. Two millilitres of butanol was added to tubes and the coloured phase was extracted by centrifugation at 3000 rpm. Absorption of the butanol phase was determined spectrophotometrically at 532 nm wavelength. Thiobarbituric acid reactive substances were expressed as micromole of MDA per litre of plasma (μmol L−1).

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Determination of antioxidant enzymes

SOD activity was measured kinetically by a method described by Sun and colleagues [12]. The principle of the method is based on the inhibition of nitro-blue tetrazolium reduction by the xanthine–xanthine oxidase system as a superoxide generator. SOD activity was expressed as units per millilitre (U mL−1).

CAT activity was measured as described previously by Aebi [13]. This method uses the change in absorbance at 240 nm at 25°C of a solution of 10 mmol H2O2 in phosphate buffer, pH 7.0. The decrease in absorbance per unit time is a measure of the CAT activity and expressed as units per litre (U L−1).

GPx activity was measured by a method described by Paglia and colleagues [14]. GPx catalyses the oxidation of glutathione. In the presence of glutathione reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized glutathione is converted to the reduced form with a concomitant oxidation of reduced NADPH to NADP. GPx activity was measured by the decrease of reduced NADPH absorbance at 340 nm and expressed as units per millilitre (U mL−1).

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Statistical analysis

Data were expressed as the mean ± SD. Differences between the three groups were analysed with the Kruskal–Wallis one-way analysis of variance (ANOVA) test. Significant differences between two groups were analysed with the Mann Whitney U-test. Changing patterns of MDA, SOD, CAT and GPx in each group were evaluated by Friedman's two-way ANOVA and differences were considered significant when P < 0.05.

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Results

There were no significant differences between groups in terms of age, weight, height, gender distribution, or tourniquet time (Table 1). Patients' baseline values of MDA, SOD, CAT and GPx were not statistically different between the groups (P > 0.05).

Table 1

Table 1

Plasma concentrations of MDA increased significantly in Group G at BTR and 5 and 20 min ATR by means of 4.5 ± 0.5, 4.7 ± 0.4 and 4.8 ± 0.2 μmol L−1, respectively, compared with the baseline value (3.3 ± 0.4 μmol L−1) (P < 0.05). In Groups S and T, significantly decreased levels of MDA were observed both at BTR (MDAS: 1.9 ± 0.4, MDAT: 2.1 ± 0.2) and 5 (MDAS: 1.8 ± 0.3, MDAT: 1.9 ± 0.5) and 20 min (MDAS: 2.0 ± 0.6, MDAT: 2.2 ± 0.5) ATR compared with the baseline value (MDAS: 3.6 ± 0.5, MDAT: 3.5 ± 0.8) (P < 0.05). There was no significant difference between Groups S and T in terms of plasma MDA levels (Fig. 1).

Figure 1.

Figure 1.

Plasma SOD and CAT concentrations decreased at BTR and 5 and 20 min ATR when compared with baseline in Group G, but these decreases were only statistically significant at 5 and 20 min ATR (P < 0.05). Although not statistically significant, SOD and CAT concentrations were slightly decreased compared to baseline in both Groups S and T (P > 0.05) (Figs 2 and 3). Plasma GPx levels were not significantly altered in any groups (P > 0.05).

Figure 2.

Figure 2.

Figure 3.

Figure 3.

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Discussion

Extremity surgery with tourniquet to provide a bloodless field is a good human model for IR injury. Postischaemic reperfusion injury is associated with the generation of ROS (H2O2, O2, OH) which damage cellular components and initiate the lipid peroxidation process. This reaction produces toxic metabolites such as MDA. Thus, MDA is often assayed as reflecting lipid peroxidation level [15,16]. The human body has a complex antioxidant defence system that includes the antioxidant enzymes such as SOD, CAT and GPx. These enzymes prevent the either initiation or propagation of free radical chain reactions [17–19].

Recently advances in the understanding of reperfusion injury and the pharmacology of antioxidants has made the interruption of reperfusion injury clinically promising. Free-radical mediated tissue injury can be limited by the use of antioxidant therapy and several studies have suggested a positive role for anti-oxidant therapy in skeletal muscle reperfusion injury [20,21]. Propofol is chemically similar to phenol-based free radical scavengers such as butylated hydroxytoluene and the endogenous antioxidant α-tocopherol (vitamin E) [7,22]. It was shown previously that propofol attenuates tourniquet induced IR injury in human beings when compared with other anaesthetics agents [9,10].

We observed a decrease in MDA levels with respect to baseline values before and after release of tourniquet in both Groups S and T with small dose continuous sedation and continuous i.v. infusion, respectively. Cheng and colleagues [10] observed small dose propofol sedation attenuates the formation of ROS in tourniquet induced IR injury compared to midazolam sedation. Our results in Group S seemed to be in line with this figure reported earlier in the literature. Also decreased MDA levels both at BTR and ATR in Group T was similar with the work of Aldemir and colleagues [23]. In these groups plasma SOD and CAT concentrations were not changed. We consider that continuous administration of propofol either small dose sedation or i.v. infusion attenuated oxidative stress, improved activities of antioxidant enzymes, particularly SOD and CAT, and decreased concentrations of MDA. This reduction may be explained by the fact that tissues below the tourniquet are saturated with propofol. Indeed, propofol is known to accumulate in biomembranes quite rapidly and may be able to boost the antioxidant defences of cells and tissues, especially in membranes [7,24]. Therefore, free-radical production and subsequent lipid peroxidation following tourniquet release might have been prevented with propofol bound to proteins or present in membranes.

We observed similar changes in MDA and antioxidant enzyme levels in Groups S and T. This may be due to the effects of both propofol and spinal anaesthesia which reduce the stress-inducing hormones, such as adrenaline, noradrenaline, and cortisol [25–27].

Although halothane offered some protective effects against reperfusion injury at the cellular level [28,29], propofol use as an induction dose only in Group G was not effective as the others. The elevation in lipid peroxides content was accompanied by a decrease in antioxidant enzyme levels in Group G. CAT activity was significantly lowered ATR, an effect that may be related to increased H2O2 production. Moreover, the reduction in CAT activity was accompanied by a concomitant decreased in SOD activity, an effect which goes in line with other investigators [30–32]. However, the GPx content, which was expected to be affected by the release of free radicals, was unchanged in all groups. According to our results this was in the same line with the literature [33].

ROS production after IR injury may bring more severe complications and represents a source of substantial morbidity and mortality in various fields of medicine. According to our results propofol use either as a small dose sedation adjunct to spinal anaesthesia or as a part of total i.v. anaesthesia may offer advantages by inhibiting lipid peroxidation and restoring antioxidant enzyme levels in case of anticipated IR injury such as would occur in extremity surgery requiring tourniquet application.

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References

1. Wakai A, Wang JH, Winter DC et al. Tourniquet-induced systemic inflammatory response in extremity surgery. J Trauma 2001; 51: 922–926.
2. Concannon MJ, Kester CG, Welsh CF et al. Patterns of free-radical production after tourniquet ischemia: implications for the hand surgeon. Plast Reconstr Surg 1992; 89: 846–852.
3. Grisham MB, Granger DN. Free radicals: reactive metabolites of oxygen as mediators of postischemic reperfusion injury. In: Martson A, Bulkley GB, Fiddian-Green RG, Haglund U, eds. Splanchnic Ischemia and Multiple Organ Failure. St Louis: Mosby, 1989: 135–144.
4. McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000; 108: 652–659.
5. Bulkley GB. Free radicals and other reactive oxygen metabolites: clinical relevance and the therapeutic efficacy of antioxidant therapy. Surgery 1993; 113: 479–483.
6. Friedl HP, Till GO, Trentz O et al. Role of oxygen radicals in tourniquet-related ischemia-reperfusion injury of human patients. Klin Wochenschr 1991; 69: 1109–1112.
7. Murphy PG, Myers DS, Davies MJ et al. The antioxidant potential of propofol (2,6-diisopropylphenol). Br J Anaesth 1992; 68: 613–618.
8. Runzer TD, Ansley DM, Godin DV et al. Tissue antioxidant capacity during anesthesia: propofol enhances in vivo red cell and tissue antioxidant capacity in a rat model. Anaesth Analg 2002; 94: 89–93.
9. Kahraman S, Kilinc K, Dal D et al. Propofol attenuates formation of lipid peroxides in tourniquet-induced ischemia-reperfusion injury. Br J Anaesth 1997; 78: 279–281.
10. Cheng YJ, Wang YP, Chien CT et al. Small dose propofol sedation attenuates the formation of reactive oxygen species in tourniquet-induced ischemia-reperfusion injury under spinal anesthesia. Anaesth Analg 2002; 94: 1617–1620.
11. Yoshioka T, Kawada K, Shimada T et al. Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am J Obstet Gynecol 1979; 135(3): 372–376.
12. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497–500.
13. Aebi H. Catalase in vitro. Method Enzymol 1984; 105: 121–126.
14. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158–169.
15. Valenzuela A. The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress. Life Sci 1991; 48: 301–309.
16. Lindsay TF, Liauw S, Romaschin AD et al. The effect of ischemia/reperfusion on adenine nucleotide metabolism and xanthine oxidase production in skeletal muscle. J Vasc Surg 1990; 12: 8–15.
17. Anaya-Prado R, Toledo-Pereyra LH, Lentsch AB et al. Ischemia/reperfusion injury. J Surg Res 2002; 105: 248–258.
18. Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1998; 255: H1269–H1275.
19. Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 1992; 119: 598–620.
20. Mohler LR, Pedowitz RA, Ohara WM et al. Effects of an antioxidant in a rabbit model of tourniquet induced skeletal muscle ischemia-reperfusion injury. J Surg Res 1996; 60: 23–28.
21. Bushell A, Klenerman L, Davies H et al. Ischemia reperfusion-induced muscle damage: protective effect of corticosteroids and antioxidants in rabbits. Acta Orthop Scand 1996; 67: 393–398.
22. Green TR, Bennett SR, Nelson VM. Specificity and properties of propofol as an antioxidant free radical scavenger. Toxicol Appl Pharmacol 1994; 129: 163–169.
23. Aldemir O, Celebi H, Cevik C et al. The effects of propofol or halothane on free radical production after tourniquet induced ischaemia-reperfusion injury during knee arthroplasty. Acta Anaesth Scand 2001; 45: 1221–1225.
24. Demiryürek AT, Cinel I, Kahraman S et al. Propofol and intralipid interact with reactive oxygen species: a chemiluminescence study. Br J Anaesth 1998; 80: 649–654.
25. Ng A, Tan SSW, Lee HS et al. Effect of propofol infusion on the endocrine response to cardiac surgery. Anaesth Intens Care 1995; 23: 543–547.
26. Erskine R, Janicki PK, Ellis P et al. Neutrophils from patients undergoing hip surgery exhibit enhanced movement under spinal anaesthesia compared with general anaesthesia. Can J Anaesth 1992; 39: 905–910.
27. Erskine R, Janicki PK, Neil G et al. Spinal anaesthesia but not general anaesthesia enhances neutrophil biocidal activity in hip arthroplasty patients. Can J Anaesth 1994; 41: 632–638.
28. Schlack W, Preckel B, Stunneck D et al. Effects of halothane, enflurane, isoflurane, sevoflurane and desflurane on myocardial reperfusion injury in the isolated rat heart. Br J Anaesth 1998; 81: 913–919.
29. Kato R, Foëx P. Myocardial protection by anaesthetic agents against ischemia-reperfusion injury: an update for anaesthesiologists. Can J Anaesth 2002; 49: 777–791.
30. Aabdallah DM, Eid NI. Possible neuroprotective effects of lecithin and α-tocopherol alone or in combination against ischemia/reperfusion insult in rat brain. J Biochem Mol Toxicol 2004; 18: 273–278.
31. Chan PH, Chu L, Fishman RA. Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia. Brain Res 1988; 439: 388–390.
32. Kinuta Y. Lipid peroxidation and changes in xanthine oxidase in cerebral ischemia. Nippon Geka Hokan 1989; 8: 59–70.
33. Zhang L, Maiorino M, Roveri A et al. Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases. Biochim Biophys Acta 1989; 1006: 140–143.
Keywords:

ANAESTHETICS INTRAVENOUS, propofol; REPERFUSION INJURY; TOURNIQUETS; ANTIOXIDANTS; LIPID PEROXIDATION; SURGERY, lower limbs

© 2007 European Society of Anaesthesiology