General anaesthesia can impair immunological defence mechanisms while inducing an inflammatory reaction in alveolar macrophages. Generalized inflammatory reactions involving the production of leucocytes release inflammatory mediators and free oxygen radicals . Damage to membrane lipids by free radicals is implied by the appearance of lipid peroxidation products during general anaesthesia [2,3], e.g. malondialdehyde. Superoxide dismutase is an important defence against such oxidation [4,5]. The effects of volatile anaesthetics, such as isoflurane, halothane, desflurane and sevoflurane on the malondialdehyde and superoxide dismutase responses have been studied, but there is no study comparing sevoflurane and desflurane in human beings. Hence, our objective was to investigate and compare the effects of sevoflurane and desflurane anaesthesia on oxidative stress and antioxidative response by determining malondialdehyde and superoxide dismutase concentrations in plasma and cells obtained from bronchoalveolar lavage fluid. The samples were obtained during laparoscopic cholecystectomy in healthy human beings.
The study was approved of by the Human Studies Review Board of the Cerrahpasa Medical Faculty of Istanbul University, and informed consent was obtained from all patients. The subjects enrolled into the study had a diagnosis of cholecystitis and comprised 40 healthy patients aged 30-60 yr, ASA I-II, undergoing elective laparoscopic cholecystectomy. Patients were allocated randomly to be anaesthetized either with sevoflurane (n = 20) or desflurane (n = 20). Exclusion criteria were: major bleeding, endocrine or immune system disease, malignant disorders, and cardiovascular, respiratory, renal or hepatic disease.
No premedication was given. Monitoring consisted of continuous measurements of the electrocardiogram, heart rate, respiratory rate, non-invasive arterial pressure (systolic, diastolic, mean), peripheral arterial saturation by pulse oximetry, end-tidal PCO2 (ETCO2), axillary temperature and inhaled anaesthetic gas concentrations (Millennia® 3500; Vital Signs, Parkway, Orlando, FL, USA). A 20-G Teflon® cannula was inserted into a vein on the back of the hand. Balanced salt solutions (0.9% NaCl, 2 mL kg−1 h−1) were given pre- and perioperatively. Anaesthesia was induced with propofol 2 mg kg−1 and fentanyl 1 μg kg−1 intravenously (i.v.), and orotracheal intubation was facilitated with cisatracurium 0.2 mg kg−1. Patients' lungs were ventilated with 40% oxygen in air at a frequency of 12 breaths min−1 and tidal volume was adjusted to maintain ETCO2 at 4.6-5.3 kPa (VT, 8 mL kg−1; PEEP, 4 cmH2O; peak airway pressure, 15-20 cmH2O). Anaesthesia was maintained either with sevoflurane at an inspiratory concentration of 2-3% (1-1.5 MAC), or desflurane 6-9% (1-1.5 MAC). A second bolus dose of fentanyl 50 μg was given immediately before skin incision. Additional boluses of fentanyl 50 μg were given if the heart rate increased by more than 30%. Intra-abdominal inflation pressures were limited to about 12 cmH2O during laparoscopic surgery.
Sample preparation, storage and assay
Blood samples (2.5 mL) were obtained before induction of anaesthesia, immediately before skin incision (about 20 min after induction) and at the end of the surgery. As our study bore no direct benefit for the patient examined, relatives were fully informed about risks of blind alveolar lavage. Rather than using the normal blind alveolar lavage procedure that instills up to 150 mL of saline, we used a reduced volume for our non-bronchoscopic blind bronchoalveolar lavage technique. Alveolar cells were obtained in this way after induction of anaesthesia and at the end of surgery, i.e. before endotracheal extubation. The broncholavage tube system was introduced blindly into the bronchial tree, the inner catheter advanced until resistance was encountered and then sterile saline 20 mL injected . Heparinized blood and broncholavaged samples were centrifuged (3000 rpm, 10 min, 0-4°C) and the supernatant fluid was immediately stored at −80°C until assayed. Lipid peroxidation was detected by the formation of malondialdehyde, which was estimated by the thiobarbituric acid method . Concentrations of malondialdehyde were calculated using an extinction coefficient of 1.56 × 105 mol cm−1; malondialdehyde was expressed as nmol mL−1. Superoxide dismutase activity was determined by the method of Sun and colleagues , which involves inhibition of nitrobluetetrazolium reduction by superoxide anions; superoxide dismutase is expressed in U mL−1.
All data are expressed as mean and standard deviation (or range), and 95% confidence intervals. Patient characteristics and biochemical data were compared between groups using the t-test, when appropriate, and by multiple variant tests (pairwise comparisons) between the groups using the least significant difference test. Within group changes were analysed using analyses of variance for repeated measures of ANOVA. Statistical power analysis was undertaken in which an 100% probability rate of detecting a increase in plasma superoxide dismutase concentration from 76% in the sevoflurane group to 51% in the desflurane group was required at the P = 0.05 level. Twenty patients would be sufficient in each group for analysis. The data were tested for normal distributions.
There were no differences between the groups in ages, weight and duration of surgery (Table 1). There were no differences between the groups over time in heart rate, arterial pressure, SPO2, ETCO2 and temperature (data not shown).
Plasma malondialdehyde concentrations increased after both anaesthetics (P < 0.001), but plasma superoxide dismutase values increased only after desflurane (P < 0.05 after induction, P < 0.05 at end of the surgery). Plasma malondialdehyde and superoxide dismutase concentrations increased more in the desflurane group than in the sevoflurane group - both after induction and after completion of surgery (P < 0.001 for malondialdehyde and superoxide dismutase after induction, P < 0.01 for superoxide dismutase at the end of surgery) (Table 2). Plasma superoxide dismutase concentrations decreased in the sevoflurane group after induction (P < 0.01) (Table 2). Malondialdehyde concentrations in the alveolar cells increased significantly in the desflurane group (P < 0.001) but not in the sevoflurane group (Table 3).
Previous human studies on this subject have not evaluated the individual contributions of such factors as mechanical ventilation of the lungs, general anaesthesia and surgical stress . We believe that each of these factors may contribute significantly to the intraoperative pulmonary inflammatory response [10-12]. We chose to study a laparoscopic procedure, because acute phase responses are significantly less than after open surgery [13,14]. The effects of sevoflurane and desflurane on the oxidative stress response during laparoscopic cholecystectomy have not previously been investigated.
Washing out of the lungs with saline or mucolytic agents for diagnostic or therapeutic purposes has proved very useful. Allaouchiche and colleagues  measured plasma and alveolar malondialdehyde concentrations during general anaesthesia in pigs anaesthetized with propofol, sevoflurane or desflurane. They found a significant increase in plasma and alveolar malondialdehyde with desflurane, but no significant changes of plasma superoxide dismutase concentrations with any agent. In vitro, halothane metabolism causes lipid peroxidation in microsomes under a variety of conditions [16,17]; sevoflurane has less effect . Mogil'nitskaia and colleagues , demonstrated raised plasma concentrations of malondialdehyde after 3 h in hypoxic rats, but no changes in plasma superoxide dismutase concentrations.
Many disease states (sepsis, burns, ischaemic-reperfusion injury, chronic obstructive pulmonary disease, hepatitis, etc.) lead to excessive lipid peroxidation and oxidative stress. Boya and colleagues  found greater superoxide dismutase concentrations in patients with chronic hepatitis. Eger and colleagues  applied desflurane 1.6 MAC or isoflurane in oxygen, or oxygen alone (control) for 2 h and 3 days in Sprague-Dawley rats, which were then sacrificed. In the desflurane group, apart from atelectasis, pulmonary injury was found. For obvious reasons, our study did not include histopathological examination of the lung.
In this study, both agents caused lipid peroxidation; plasma and alveolar cell malondialdehyde concentrations were greater with desflurane than sevoflurane, after induction and end of the study. Thus desflurane caused more lipid peroxidation than sevoflurane. Inflammation was initiated following endotracheal intubation and enhanced by the addition of inhalational anaesthetic agents [11,12], particularly desflurane. As our sampling times were earlier than in other studies, elevated plasma and alveolar cell malondialdehyde concentrations were seen earlier, especially in the desflurane group. In this group, superoxide dismutase concentrations in both plasma and the alveolar cells were increased. However, plasma superoxide dismutase concentrations were only significantly higher in the desflurane group. Although these increases do not have any clinical implications, the raised plasma malondialdehyde concentrations suggest that this may be regarded as a healthy response to oxidative stress. In the sevoflurane group superoxide dismutase concentrations were decreased after induction; however, we are unable to explain the reason for this.
We were unable to find the normal ranges for malondialdehyde and superoxide dismutase in previous reports. Since our study was conducted on healthy subjects, we accepted the initial values as the control values. Initial malondialdehyde and superoxide dismutase values were all very similar. We are not aware of any other studies in healthy human beings with which to compare our results. In previous studies of lipid peroxidation during inhalation anaesthesia, the subjects were either animals or patients with chronic disease, which may explain the different results. In conclusion, we found that desflurane appears to cause more systemic and regional lipid peroxidation than sevoflurane during laparoscopic cholecystectomy in healthy human beings. However, further clinical studies are required to investigate the lipid peroxidation effects of desflurane.
1. Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med
2. Kotani N, Lin CY, Wang JS, et al.
Loss of alveolar macrophages during anaesthesia and operation in humans. Anesth Analg
3. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants and human disease: where are we now? J Lab Clin Med
4. Mercer RR, Russell ML, Roggli VL, Crapo JD. Cell number and distribution in human and rat airways. Am J Respir Cell Mol Biol
5. McCord JM. The evaluation of free radicals and oxidative stress. Am J Med
6. Rouby JJ, Rossignon MD, Nicholas MH. A prospective study of protected bronchoalveolar lavage in the diagnosis of nasocomial pneumonia. Anesthesiology
7. Angel MF, Ramasastry SS, Swartz WM, et al.
The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion. Plastic Reconstruct Surg
8. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem
9. Haga Y, Beppu T, Doi K, et al.
Systemic inflammatory response syndrome and organ dysfunction following gastrointestinal surgery. Crit Care Med
10. Suter PM, Ricov B. Cytokines and lung injury. In: Marini JJ, Evans TW, eds. Acute Lung Injury.
Berlin, Germany: Springer-Verlag, 1998: 41-53.
11. Wrigge H, Zinserling J, Stüber F, et al.
Effects of mechanical ventilation on release of cytokines into systemic circulation in patients with normal pulmonary function. Anesthesiology
12. Kotani N, Takahaski S, Sessler DI, et al.
Volatile anesthetics augment expression of proinflammatory cytokines in rat alveolar macrophages during mechanical ventilation. Anesthesiology
13. Douglas RG, Shaw JHF. Metabolic response to sepsis and trauma. Br J Surg
14. Stahl W. Acute phase protein response to tissue injury. Crit Care Med
15. Allaouchiche B, Debon R, Goudable J, Chassard D, Duflo F. Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg
16. De Groot H, Noll T. Halothane-induced lipid peroxidation and glucose-6-phosphatase inactivation in microsomes under hypoxic conditions. Anesthesiology
17. National Research Council (USA). In: Bunker JP, Forest WH, Mosteller H, et al. National Halothane Study: A Study of the Possible Association between Halothane Anesthesia and Postoperative Hepatic Necrosis.
Washington DC, USA: Government Printing Office, 1969.
18. Frink E, Brown B. Sevoflurane. Anaesth Pharmacol Rev
19. Mogil'nitskaia LV, Prokof'ev VN, An F, Zhogolev VV. The effect of hypoxia on membrane status and lipid peroxidation in rat lungs and blood. Vopr Med Khim
20. Boya P, de la Pena A, Beloqui O, et al.
Antioxidant status and glutathione metabolism in prepheral blood mononuclear cells from patients with chronic hepatitis C. J Hepatol
21. Eger II El, Johnson BH, Ferrell LD. Comparison of the toxicity of I-653 and isoflurane in rats: a test of the effect of repeated anesthesia and use of dry soda lime. Anesth Analg
Keywords:© 2004 European Society of Anaesthesiology
ALDEHYDES, malondialdehyde, thiobarbituric acid reactive substances; ANAESTHETICS INHALATION, desflurane, sevoflurane; ENERGY METABOLISM, oxidation-reduction, lipid peroxidation; ENZYMES, oxidoreductases, superoxide dismutase; INVESTIGATIVE TECHNIQUES, irrigation, bronchoalveolar lavage