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 . Postischaemic reperfusion injury is associated with the generation of reactive oxygen species (ROS) which damage cellular components and initiate the lipid peroxidation process . 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 . 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) . 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.
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.
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 . 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).
Determination of antioxidant enzymes
SOD activity was measured kinetically by a method described by Sun and colleagues . 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 . 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 . 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).
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.
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).
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).
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).
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, O−2, 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  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 . 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 .
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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Keywords:© 2007 European Society of Anaesthesiology
ANAESTHETICS INTRAVENOUS, propofol; REPERFUSION INJURY; TOURNIQUETS; ANTIOXIDANTS; LIPID PEROXIDATION; SURGERY, lower limbs