With the routine use of muscle relaxants, the control of sympathetic responses induced by surgical incision represents one of the most important end-points for assessing the depth of anaesthesia  and no individual anaesthetic drug is commonly used alone to provide all the necessary components of general anaesthesia.
It has been clearly demonstrated that fentanyl reduces the minimum alveolar concentration (MAC) of sevoflurane required to abolish consciousness, reduce the response to intubation and blunt the sympathetic response to skin incision [2-4]. Remifentanil is a new selective μ opioid receptor agonist providing intense analgesia of rapid onset and ultra-short duration . Remifentanil has been demonstrated to be effective and safe in preventing sympathetic responses induced by tracheal intubation and other surgical stimuli [6-8]. Due to its unique pharmacokinetic and pharmacodynamic profile, remifentanil is ideally suited for continuous intravenous (i.v.) infusion [5,6], while the use of a target-controlled infusion (TCI) using a computer-driven infusion device has been demonstrated to be more effective in maintaining cardiovascular stability than a traditional weight-adjusted infusion . The ability of nitrous oxide to reduce the requirement for other anaesthetic agents has been well described in previous investigations. The MAC of sevoflurane to block the adrenergic response to surgical incision in female has been recently reported ; however, there is no information on the contribution of nitrous oxide in reducing the MAC of sevoflurane in female in the presence of different concentrations of remifentanil. We therefore conducted this prospective, randomized, double-blind study to determine the effects of adding 60% nitrous oxide on the MAC of sevoflurane in the presence of two different target-controlled effect-site concentrations of remifentanil (1 and 3 ng mL−1) in female.
After obtaining Institutional Ethics Committee approval and each patient's written informed consent, 102 female patients, aged 20-50 yr, ASA I, scheduled for elective abdominal surgery requiring skin incision were prospectively enrolled. Patients undergoing laparoscopic procedures, obese patients (body mass index, BMI >30), patients with a history of cardiac, pulmonary or renal diseases, drug or alcohol abuse, or current use of any medications affecting the cardiovascular system or blocking the adrenergic responses to surgical incision were excluded. No local anaesthetic agents, atropine, epinephrine or other vasoactive medications were used before the skin incision.
Patients fasted for 8 h before surgery and received no premedication. After arrival in the operating theatre an 18-G i.v. cannula was inserted in the forearm, and 10 mL kg−1 Ringer's lactate solution was infused. Standard monitoring was used throughout the study, including non-invasive arterial pressure (Dinamap 1846SX; Critikon, FL, USA), electrocardiography, heart rate (HR; Lead II), and pulse oximetry.
Using a computer-generated sequence of numbers, patients were randomly allocated to one of two groups: 60% nitrous oxide in oxygen mixture (Group N, n = 49) or 40% oxygen in air mixture (Group A, n = 53). In each of the two groups, patients were further randomized to received a TCI of remifentanil set to maintain an effect-site concentration of either 1 ng mL−1 (Group N1, n = 27; and Group A1, n = 30), or 3 ng mL−1 (Group N3, n = 22; and Group A3, n = 23).
General anaesthesia was induced with i.v. propofol (2 mg kg−1) and a TCI of remifentanil set at 4 ng mL−1 for intubation, which was facilitated with cisatracurium besilate 0.2 mg kg−1. The lungs were ventilated with sevoflurane combined with the designated mixture. According to the patients group, the TCI of remifentanil was set at the desired concentration ensuring an adequate equilibration time between plasma and effect site based upon the very short equilibration-time between plasma and effect site (KeO) [5,9].
The first patient assigned to all groups received 1.5 MAC of sevoflurane adjusted for age (3%) . The target end-tidal concentration of sevoflurane was maintained stable for up to 20 min before surgical incision. Inspired and end-tidal oxygen, carbon dioxide, and sevoflurane concentrations were measured continuously using an infrared gas analyser (RGM 5250, Ohmeda) calibrated before each case according to the manufacturer instructions. Ventilation was mechanically controlled using a Cato-Dräger anaesthesia workstation (Dräger, Lubeck, Germany) set to maintain an end-tidal partial pressure of carbon dioxide ranging between 32 and 35 mmHg. Fresh gas flow was set at 10 L min−1 flow to rapidly reach and maintain the designated end-tidal concentration of sevoflurane.
Remifentanil was administered using a pharmacokinetic model-driven computer-assisted continuous infusion system to achieve and maintain constant target plasma concentrations . The system consisted of an Acer TravelMate 518TX computer connected to a Graseby 3500 infusion pump (Sims Graseby Limited, Watford, Herts, UK) [9,12,13] using the Rugloop software (designed by Tom De Smet and Michel Struys, Department of Anesthesia, University Hospital, Ghent, Belgium). The pharmacokinetic parameters used in the computer-assisted continuous infusion for administration of remifentanil were based on the model described by Minto and colleagues [13,14].
HR and mean arterial pressure determined by oscillometry were recorded before induction of anaesthesia, 2 min and 1 min before skin incision, at skin incision, and then at 1-min intervals over the first 5 min after surgical incision. The pre-incision value was defined as the mean value of the 2- and 1-min measurements. If arterial pressure decreased before skin incision to a level that required the administration of a vasoactive agent (mean arterial pressure <50 mmHg) the patient was withdrawn from the study, and the same concentration of sevoflurane repeated with the following case. At the first postoperative day visit patients were also questioned about any recall of intraoperative events. The anaesthesiologist recording cardiovascular parameters and determining the positive-negative response to surgical incision was blinded to patient grouping.
The MAC of sevoflurane was determined using a modified up-and-down sequential allocation technique [15-17]. The response of the preceding patient determined the concentration of sevoflurane given to succeeding patients in each group. If the response of the preceding patient in that group was positive (an increase of either HR or blood pressure (BP) ≥15% above the mean of the values measured during the 2 min before skin incision), the end-tidal concentration given to the next patient was increased by 0.25 MAC (0.5%). If the response was negative (neither HR nor BP increased ≥15% above the mean of the values measured during the 2 min before skin incision), the end-tidal concentration of sevoflurane given to the next patient was decreased by 0.25 MAC (0.5%). According to the modified up-and-down method, the sequence is formed by two stages: the first stage consists of an original up-and-down sequence on the predetermined equally spaced test levels until three positive-negative deflections are observed. The second stage consists of reducing the initial test space, restarting the up-and-down sequence at the nearest level to the average and continuing the experiment at the next higher or next lower level according to the response type on the reduced test space. According to this modified up-and-down method, the initial test space was halved to 0.1 MAC  (0.2%) for an a priori number of independent positive-negative up-and-down deflections of four with the new reduced test space. Reducing the test space increases the precision of the final estimator, reducing the mean squared error under normal tolerance distribution and has been shown to be substantially better than the original method when the initial test space is relatively wide and the initial dose is away from the final median dose. The mean (95% confidence interval, CI95) of the MAC of sevoflurane was calculated from the mid-points of paired concentrations from consecutive patients, in which a negative response was followed by a positive one after the initial test space was reduced according to the modified up-and-down method [15-17]. The data were also analysed using a logistic regression model to estimate the effective sevoflurane concentration needed for blockade of cardiovascular responses in 50% and 95% of patients (ED50 and ED95, respectively).
Statistical analysis was performed using the program Statistica 5.1 (StatSoft Italia, Vigonza, Padova, Italy). A two-way analysis of variance for repeated measures was used to analyse changes over time in cardiovascular variables. Scheffé's test was used for post hoc comparisons. With all pooled data obtained from the up-and-down sequence in the four groups we also calculated the ED50 and ED95 using a probit transformation and a logistic regression analysis. A value of P ≤5% was considered statistically significant. Data are presented as mean (±SD) and CI95.
A total of 102 female patients completed the study. No differences in patient characteristics, preoperative HR and mean arterial pressure were reported among the groups. The comparison of pre- and post-skin incision HR and mean arterial pressure within and between groups showed the post-skin incision mean arterial pressure between Groups A3 and N3 to be significantly different (Table 1). Three patients in Group N3 and one patient in Group A1 were withdrawn from the study for hypotension requiring vasoactive agents. No patient recalled any event occurring during the study at the 24 h postoperative visit.
Figures 1-4 show individual responses to skin incision according to the up-and-down sequence. The MAC of sevoflurane that suppressed the sympathetic response to surgical incision (MACBAR) in 50% of subjects (CI95) receiving a target-effect-site concentration of remifentanil of 1 and 3 ng mL−1 was 3.9% (CI95: 3.7-4.2%) and 0.36% (CI95: 0.24-0.47%) (P < 0.001). The addition of 60% nitrous oxide reduced the MACBAR of sevoflurane to 1.2% (CI95: 0.9-1.3%) and 0.18% (CI95: 0.1-0.3%), in the presence of 1 and 3 ng mL−1, respectively (P < 0.05).
The ED50 and ED95 calculated from logistic regression analysis in patients not receiving nitrous oxide were 3.5% ± 0.3% and 10.7% ± 0.3% when using 1 ng mL−1 remifentanil (1.7 and 5.0 MAC, respectively) and 0.4% ± 0.2% and 1.5% ± 0.2% when using 3 ng mL−1 remifentanil (0.18 and 0.75 MAC, respectively). The ED50 and ED95 calculated from logistic regression analysis in the presence of 60% nitrous oxide were 1.6% ± 0.2% and 1.9% ± 0.3% when using 1 ng mL−1 remifentanil (1.3 and 1.5 MAC, respectively), and 0.27% ± 0.05% and 0.29% ± 0.2% in the group receiving 3 ng mL−1 remifentanil (0.68 and 0.7 MAC, respectively). The ED50 values calculated from logistic regression analysis did not differ significantly from the MACBAR value obtained with the Dixon method.
The sympathetic response to surgical incision is a clinically relevant end-point for assessing depth of anaesthesia. Roizen and colleagues  described the property of inhalational anaesthetics of blunting haemodynamic responses to skin incision, and defined the MACBAR as the concentration of a volatile anaesthetic agent needed to block the sympathetic responses to surgical incision in 50% of patients. The effect of nitrous oxide on the MACBAR of sevoflurane have been clearly described by Nickalls and Mapleson in a recent study ; however, in modern balanced anaesthesia, volatile anaesthetics are usually combined with i.v. opioids and little information was available on the effects of nitrous oxide on the MACBAR of sevoflurane used in combination with the new opioid agent, remifentanil. The main finding of this prospective, randomized, double-blind study is that adding 60% nitrous oxide reduces the MACBAR of sevoflurane by 70% when used with 1 ng mL−1 remifentanil concentration and 50% when used with up to 3 ng mL−1.
In a normal population of patients Katoh and Ikeda  reported a MACBAR value of sevoflurane without nitrous oxide as high as 4.15%, which was reduced by 57% by the addition of 1 ng mL−1 fentanyl. In the present investigation we enrolled only female, and did not measure the MACBAR of sevoflurane with and without nitrous oxide in the absence of any opioid agent. However, in a similar population, Ura and colleagues  reported a MACBAR of sevoflurane without any opioid of about 8%; accordingly, the addition of 1 ng mL−1 resulted in a nearly 50% decrease of the MACBAR of sevoflurane. Katoh and Ikeda  also reported on the effects of 66% nitrous oxide on the MACBAR of sevoflurane in combination with a fentanyl plasma concentration of 1 and 3 ng mL−1 reporting values of 1.2% and 0.2%, respectively. In agreement with Ura and colleagues , the big difference observed in the present investigation could be explained by both the different statistical approach we used, and the different patient population. In Katoh's study, the concentration of fentanyl reducing the MACBAR of sevoflurane by 99% was 4.4 ng mL−1 . This finding is in agreement with results of the present investigation in which a target-controlled concentration of remifentanil of 3 ng mL−1 resulted in a 95% reduction of the MACBAR. Similarly, findings with other inhalational agents have also been reported by other authors .
The MACBAR of sevoflurane determined in patients receiving the 3 ng mL−1 concentration of remifentanil was very low, much lower than the MACAwake reported in previous investigations . These results are in agreement with those reported with similar concentrations of fentanyl , and could potentially result in an inadequate level of hypnosis in these patients. Despite the use of very low concentrations of sevoflurane at skin incision in these patients, none reported explicit recall at the postoperative follow-up visit. Nonetheless, to minimize the risks of awareness in patients receiving the 3 ng mL−1 concentration of remifentanil we stopped patient enrolment as soon as we had the minimum positive-negative deflections in the up-and-down sequence. However, it must be pointed out that even with small doses of opioids, patients can be aware of intraoperative events despite the lack of changes in cardiovascular parameters [2,20,21], and this may suggest the use of specific monitors to evaluate the degree of hypnosis during balanced anaesthesia with sevoflurane and potent opioids like remifentanil .
To provide adequate protection from the stress response induced by tracheal intubation, all patients received a TCI of remifentanil set at 4 ng mL−1 before tracheal intubation, and this could be a potential shortcoming of the study. However, the remifentanil infusion was set at the desired target-controlled concentration immediately after tracheal intubation while the target concentration of sevoflurane was maintained stable for >20 min before skin incision. Based on remifentanil's pharmacokinetic and pharmacodynamic properties, these times allow equilibration of the calculated effect-site concentration at the point of MACBAR determination.
The lack of direct determination of plasma concentrations of remifentanil was another shortcoming of the study. However, the pharmacokinetic model we used to achieve, and maintain stable plasma concentration of remifentanil has been demonstrated to be sufficiently accurate in predicting plasma and effect-site concentrations of remifentanil [9,13,14]. The use of a TCI of remifentanil still requires complex systems including a computer to control an infusion pump. However, for practical purposes, when using conventional weight-adjusted administration similar results can be achieved in daily practice infusing a 1 μg kg−1 bolus during a 60-s period followed by a continuous infusion of either 0.03 μg kg−1 min−1 for the 1 ng mL−1 concentration or 0.12 μg kg−1 min−1 for the 3 ng mL−1 concentration.
In conclusion, this prospective, randomized study has shown that adding 60% nitrous oxide reduces the MACBAR of sevoflurane by 70% when used in combination with 1 ng mL−1 remifentanil and 50% when used with 3 ng mL−1 remifentanil.
This study was supported by the Vita-Salute University of Milano.
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