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Accumulation of S(+) enantiomer in human beings after general anaesthesia with isoflurane racemate

Haeberle, H. A.*; Wahl, H. G.; Jakubetz, H.; Krause, H.*; Schmidt, R.; Schurig, V.; Dieterich, H.-J.*

European Journal of Anaesthesiology: September 2002 - Volume 19 - Issue 9 - p 641-646
Original Article
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Background and objective: Isoflurane is a chiral, volatile anaesthetic with low metabolic rate (0.17%) that is routinely administered in its racemic form. Knowledge about the distribution of the enantiomers in human beings may give some important information about the understanding of the mechanisms of volatile anaesthetics.

Methods: Blood samples were drawn immediately after tracheal extubation and daily up to 8 days post-operatively from patients undergoing general anaesthesia with isoflurane racemate. The enantiomer enrichment of isoflurane was determined by headspace gas chromatography-mass spectrometry.

Results: At all time points, there was a statistically significant accumulation of the S(+) enantiomer in blood, especially at days 2 (52.01%) and 7 (52.1%). Separate analysis of obese patients or in a small group of patients with co-existing lung disease did not show any difference to the total population. In addition, duration of anaesthesia did not influence the enantiomer concentrations.

Conclusions: We suggest that a slower association and dissociation rate is responsible for the S(+) enrichment.

Tuehingen University Hospital, *Department of Anaesthesiology and Intensive Care Unit,Department of Internal Medicine andInstitute for Organic Chemistry, Tuebingen, Germany

Correspondence to: Helene Haeberle, Department of Anaesthesiology and Intensive Care Unit, Tuebingen University Hospital, Hoppe-Seyler-Str. 03, D-72076 Tuebingen, Germany. E-mail: helene.haeberle@uni-tuebingen.de; Tel: +49 7071 29 86622; Fax: +49 7071 29 5533

Accepted for publication September 2001 EJA 269

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Introduction

Recent studies considering the stereoisomerism of drugs used in anaesthesia help us to understand the mode of action of anaesthetic agents. Stereoisomers are molecules that have the same chemical molecular formula and structure but a different configuration. These structures exist in two different non-superimposable mirror-image forms known as enantiomers or (R)- and (S)- isomers from the Latin words rectus and sinister. The main physical difference between these two is their ability to rotate plane-polarized light in opposite directions. Therefore the nomenclature for enantiomers specifies the rotation as (+)- and (−)-, depending on the direction of rotation of polarized light by the solution in specified conditions [1].

The kinetics and distribution, as well as the pharmacological profile, of enantiomers throw light on the mode of action of anaesthetic agents. The stereoselectivity of inhalational agents is modest and not yet clearly defined. This may be because the action of these agents is not completely clear. On the other hand, the kinetics of the enantiomers may give some information about their interaction. One example is isoflurane, a commonly used potent and safe volatile anaesthetic with a low metabolic rate (0.17%) [2]. Isoflurane (C3H2OCLF5) is a chiral molecule that exists in two non-superimposable forms, the S(+) and the R(−) enantiomer (Fig. 1). For routine clinical practice, isoflurane is applied as a racemate (50% S(+)/50% R(−)) [3]. In vitro and in vivo data from animal experiments suggest that there are differences between the anaesthetic potency and pharmacokinetic behaviour of the two enantiomers. Anaesthesia with S(+)-isoflurane is significantly more potent in mouse [4]. In rats, MAC was 17% lower for the S(+)-isoflurane compared with the R(−)-isoflurane enantiomer or the racemate. Furthermore, there is also a greater anaesthetic potency for S(+)-isoflurane (+23%) in rats [5,6]: the side-effects do not appear to be influenced by the stereoselectivity [7,8].

Figure 1

Figure 1

Additional information about the effects and distribution of the enantiomers in human beings after anaesthesia may improve our understanding of the mechanisms of these volatile anaesthetics, which is at present not clear.

The determination of the isoflurane enantiomers is not straightforward and was introduced by our group, showing how flow conditions are essential for a sensitive and specific method to obtain reproducible data [9]. In the present study, we measured the isoflurane enantiomers in the blood of patients undergoing general anaesthesia with isoflurane. The anaesthesia was maintained with the isoflurane racemate and the amount of the enantiomers was quantified immediately after anaesthesia and every day for up to 8 days postoperatively.

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Methods

Anaesthesia

All patients received midazolam as oral premedication. The induction of anaesthesia was performed according to the clinical routine: thiopental (n = 23), etomidate (4), and propofol (14) were used in combination with fentanyl (1), sufentanil (23), alfentanil (4) or remifentanil (13). Rocuronium (n = 37) or mivacurium (2) were administered intravenously (i.v.) to facilitate endotracheal intubation and maintain relaxation. In two patients, succinylcholine was used for rapid intubation.

Anaesthesia was maintained as 'balanced anaesthesia' based on the use of opioids with isoflurane (Forene®; Abbott GmbH, Wiesbaden, Germany) 'on top'. The patients' lungs were ventilated via a rebreathing circuit system (Cicero EM®; Dräger Werke AG, Lübeck, Germany) at normal end-tidal PCO2. The fresh gas flow was changed to 70% N2O/30% O2 after induction with 100% oxygen. Isoflurane was added to a final end-tidal concentration of 0.8 ± 0.2 vol% in the low flow state. At the end of the procedure, oxygenation was returned to 100% oxygen until tracheal extubation.

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Isoflurane analysis

Blood samples were drawn immediately after tracheal extubation and daily for the following 8 days by venous puncture (K-EDTA Monovette®; Sarstedt GmbH, Nümbrecht, Germany). For the measurement of the enantiomers, blood samples were transferred into headspace sample vials (2 mL GC Vials®; Klaus Ziemer GmbH, Mannheim, Germany) and stored frozen at −80°C until analysis was performed.

A multipurpose-sampler (Gerstel® MPS; Gerstel GmbH, Mülheim/Ruhr Germany) was used for the GC-MS analysis (HP 5890/5971 A; Hewlett Packard, Waldbronn, Germany). Headspace sampling with a volume-, temperature- and speed-controlled gas-tight syringe was combined with a temperature-controlled cold-injection system (CIS3®, Gerstel GmbH). Regular 2 mL GC-vials filled with 1 mL blood were equilibrated for 10 min at 70°C at the preheating station before injection. The syringe was also heated to 70°C to prevent condensation and memory effect. Injected headspace volume was 500 μL for blood samples drawn at the end of anaesthesia and 100 μL for all other samples performed by direct thermodesorption gas chromatography-mass spectrometry (TDS 2 and CIS 3®; Gerstel GmbH, Germany; HP 5890 series II®; HP 597®; Hewlett Packard, Waldbronn, Germany). Thermodesorption was achieved by raising the TDS temperature up to 220°C at 60°C min−1, kept for 10 min. CIS temperature was held at −150°C for 15 s and then heated to 310°C at 12°C s−1. During injection and cold trapping, the splitter valve was closed for 30 s.

The temperature of the detector was held at 280°C and the oven temperature was 33°C isothermally. Helium was used as the carrier gas (60 kPa column head pressure).

The capillary column (30 m × 0.25 mm × 0.28 μm) was coated with octakis (3-O-butanoyl-2,6-di-O-pentyl)-γ-cyclodextrin (Lipodex E) 30% (w/w) dissolved in PS 255 (Gelest, ABCR GmbH & Co., Karlsruhe, Germany) and connected to a retention gap. Preparation of coating of the cyclodextrin derivate is described in [9]. This special GC column with a chiral phase led to the separation of the two enantiomers. Each individual peak could then be quantified and the relative amount of S(+) and R(−) calculated. Quantification was made in the single-ion detection mode with ions m/z 51, 117 and 149.

The fraction of the enantiomers is described as percentage of total isoflurane concentration: 100% = % (S(+) enantiomer) + % (R(−) enantiomer). The intra-assay coefficient of variation for samples in the concentration ranged from 0.5 to 600 nmol mL−1 was determined as 0.9% (n = 27).

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Statistical analysis

Statistical analysis was carried out using the SigmaStat® 6.0 program (Jandel Corp., San Rafael, CA, USA). Statistic analysis of enantiomer enrichment at the different time points compared with the racemic control was made by Kruskal-Wallis one-way analysis of variance on Rank's including Dunn's method for multiple comparison. In addition, for the analysis of the subgroups of patients, the Friedman repeated measures analysis of variance on ranks was performed.

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Results

After approval of the local Ethics Committee and with written informed consent, 41 patients (of whom 25 were female) were included in the study. Patient ages were 60 ± 14 yr, height 166 ± 9 cm and body weight 73 ± 14 kg (means ± SD). The patients were ASA I-III and underwent different operative procedures with a mean duration of 194 ± 97 min (Table 1). Patients <18 yr, with mental disease or patients who were scheduled for ambulatory procedures were excluded from the study.

Table 1

Table 1

The enantiomer ratio of the vaporized isoflurane racemate was determined to be 50% ± 0.1%. The isoflurane enantiomer distribution was analysed after tracheal extubation and in the following 8 postoperative days. Six patients refused further blood drawing after 6 days and three patients after 7 days. Toward the end of the 8 days, the isoflurane concentration of some samples was below the detection limit.

Immediately after tracheal extubation, there was a shift from the applied anaesthetic isoflurane racemate with 50% S(+) and 50% R(−) towards an increase of S(+) enantiomer (Fig. 2 and Table 2). Concentrations of enantiomer enrichment were significant elevated (P < 0.05) compared with the isoflurane racemate blood control samples at each time point investigated (Kruskal-Wallis one-way analysis of variance on Ranks, Dunn's method for multiple comparison). Based on the median, the most prominent S(+) enantiomer enrichment was detected 2 days postoperatively (P < 0.001) (Mann-Whitney rank sum test).

Figure 2

Figure 2

Table 2

Table 2

Patients were categorized into subgroups according to their body mass index (BMI), duration of anaesthesia and pre-existing lung disease (Table 3). None of these factors had any significant influence on the shift toward accumulation of S(+) enantiomer of isoflurane (Figs 3 and 4), although in obese patients the fraction of S(+) enantiomer showed a slight tendency to exceed the fraction found in non-obese patients. However, the number of patients was small.

Table 3

Table 3

Figure 3

Figure 3

Figure 4

Figure 4

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Discussion

In the last decade, the interest in stereoisomerism has increased mainly due the fact that the activity of enantiomers had practical significance, as in the case of ketamine. Fluorinated inhalational agents such as halothane, enflurane and isoflurane are also chiral compounds. Although the effect of the enantiomers, especially of isoflurane, are controversial, it seems that the S(+) enantiomer is more potent in different experimental models. The reason for the missing data in human beings was because the measurement of the enantiomers was not yet established. In this study, we applied a new method to determine isoflurane enantiomers in blood samples of patients. The method was developed by the co-authors, which defined the best conditions to obtain reproducible measurements [9]. Before the present study, the kinetics of isoflurane enantiomers in human beings after application of the racemate was not known. Through the improvement of the detection method isoflurane enantiomers in blood samples were detectable up to 8 days after anaesthesia. We demonstrated a statistically significant enrichment of the S(+) enantiomer in blood samples, especially 2 and 7 days after anaesthesia performed with isoflurane racemate containing 50% S(+) and 50% R(−) enantiomers. However, the mechanism for the enrichment has not been elucidated.

The distribution of volatile anaesthetics into different tissues depends on uptake and arterial concentration of the volatile anaesthetic, perfusion of the tissue and the partition coefficient into different compartments. The two enantiomers of the vaporized racemate may have different kinetic properties during uptake and redistribution. This might explain the shift from a 50% ratio in the administrated racemate towards the enrichment of the S(+) enantiomer in the blood. Recent studies described an enrichment of S(+) enantiomers in brain tissue in rats after intravenous application of isoflurane although the differences were not significant different between S(+) and R(−) enantiomers [6]. Separate isotopic labelling of the two enantiomers may give more information about the pharmacokinetics. However, pure radioactive labelled isoflurane is not available for clinical use and its application in human beings might raise ethical and legal questions. The distribution could also be monitored by the use of fluorine nuclear magnetic resonance spectrometry (19F-NMR) after application of a pure preparation of each single enantiomer [10]. Up to now, production of such a quantity of pure enantiomers has not been considered due to the high cost.

One explanation of our results might be the different binding kinetics of the two enantiomers. It is controversial whether lipids or proteins of the membrane are the primary target of volatile anaesthetics. However, previous studies have shown that anaesthetics act on membrane proteins rather than on lipids [11]. Franks and Lieb [11] reported that anaesthetics, including isoflurane, activate potassium channels, which silence the cell by hyperpolarization. The S(+) isomer of isoflurane was about twice as effective on this potassium channel as the R(−) isomer. Furthermore, several drugs, including isoflurane, were shown to have a stereoselective effect on the γ-aminobutyric acid type A (GABAA) receptor, an inhibitory receptor-channel complex. Binding of benzodiazepines or volatile anaesthetics to this receptor increases the membrane permeability [12-14]. S(+)-isoflurane enantiomer was more potent in augmenting the flunitrazepam binding to this receptor [15]. It increased the potency and efficacy of GABA and increased the GABA-gated flux significantly more than the R(−) enantiomer [16]. Perhaps this may be the reason why the S(+) enantiomer is more potent than R(−) enantiomer in mice [4].

Our results may also reflect the different binding motives of the enantiomers: the S(+) enantiomer might bind longer to the receptor than the R(−) enantiomer, which has its highest enrichment already after recovery. The accumulation of the S(+) enantiomer may be due to the release of S(+)-isoflurane of its binding site or by its redistribution. Only one study has determined the binding kinetics of isoflurane enantiomers on bovine serum albumin, which contains saturable binding sites for isoflurane. This study using the 19F-NMR spectroscopy [17] reported no stereoselective difference in the fraction of bound isoflurane enantiomers or in their dissociation coefficient. However, the two isoflurane enantiomers showed different dynamics; S(+) enantiomer binds with a slower association and dissociation rate compared with the R(−) enantiomer. This reflects the dynamics of the S(+) enantiomer shown in this study.

We assigned the patients included in the study into groups with low (BMI < 26 kg m−2), medium (26 < BMI > 30 kg m−2) or high (BMI > 30 kg m−2) body fat mass or short (<2 h), medium (2-3 h) or long duration of anaesthesia (>3 h). The different grades of obesity in the patients had only a small influence on the increase of the S(+) enantiomer fraction. This is supported by experimental data that show no difference in the solubility of S(+) and R(−) enantiomers in lipid bilayers [18]. The increase in the S(+)-isoflurane fraction was also independent of the duration of anaesthesia. However, the data were analysed retrospectively and the patients were not selected specifically with regard to the body weight before the study.

Since the uptake of isoflurane racemate could be influenced by lung disease, we analysed subgroups with such pre-existing diseases (Table 3). In the small number of patients examined, we found no differences. However, the number of patients was very small. Further data were also analysed retrospectively and we did not investigate the enantiomer concentration specifically in one defined group of lung disease. Determination of enantiomer enrichment in a defined population of patients with lung disease may give more detailed information about the issue.

In conclusion, we suggest that the S(+) and R(−) enantiomer have different kinetics. Further investigations with labelled isoflurane enantiomers may help to unravel the reason for the enrichment of the S(+) enantiomer in humans after isoflurane anaesthesia and provide information about the molecular mechanism of isoflurane.

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Acknowledgements

Special thanks to Dr Carl Caflisch, UTMB Galveston, for copyediting the manuscript and to Professor Dietz, Tuebingen, for supporting the statistical analysis.

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Keywords:

ANAESTHETICS; INHALATION; isoflurane; ISOMERISM; stereoisomerism; MOLECULAR STRUCTURE

© 2002 European Academy of Anaesthesiology