Fluorinated volatile anaesthetics, such as isoflurane, are stereoisomeric molecules used in clinical practice as a racemate. Chiral molecules 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 enantiomers differ in 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 or in accordance to the chemical structure (R) and (S) .
Previous studies have elaborated differences between isoflurane enantiomers in vitro and in vivo. Isoflurane acts stereospecifically on γ-aminobutyric acid type A receptor  and others [3-5]. S(+) is more potent in inhibiting [3H]SR 95531 (GABA receptor antagonist) and as enhancer of [3H] muscimol binding (GABA receptor agonist) [6-8]. In binding to Ca2+ channels, on the other side there was no difference between the individual enantiomers or the racemate, or both . Several studies demonstrated a longer sleeping time and higher potency [6-10] of S(+) isoflurane enantiomer whereas Eger and colleagues did not find any difference in minimal alveolar concentration (MAC) values of the two isoflurane enantiomers .
Therefore, the stereoselective action of inhalation agents is not yet completely defined. One reason may be that detailed mechanisms of volatile anaesthetics are still under investigation. Furthermore, isoflurane enantiomers act on several receptors/channels differently. In addition, the definition of binding sites or distribution in tissues is difficult. However, since the analysis of binding properties is very troublesome, dissociation of the enantiomers from their binding sites or their kinetics may give some insights in certain action properties of these molecules.
Previously our group has introduced a sensitive and specific method to measure isoflurane enantiomers in blood . We were able to show a statistically significant increase of the S(+) enantiomer in blood samples, especially at 3 days after surgery . Since isoflurane is eliminated mostly through the lungs , it would be informative to compare enantiomer concentrations in blood samples with breath samples. Therefore, our group developed a method to determine isoflurane enantiomers in breath samples. In this new, independent study we determined kinetics of isoflurane enantiomers parallel in breath and in blood samples after operation from patients undergoing routine surgeries including application of racemic isoflurane.
After approval of the local Ethics Committee and written informal consent, the induction of anaesthesia was performed according to the clinical routine: thiopental (n = 43) or propofol (n = 3) were used in combination with sufentanil (n = 46). Rocuronium (n = 46) was administered intravenously (i.v.) to facilitate endotracheal intubation and to maintain relaxation; succinylcholine was used in two patients to facilitate rapid induction. Anaesthesia was maintained by isoflurane (Forene®; Abbott GmbH, Wiesbaden, Germany) in combination with opioids. The patients were ventilated via a rebreathing system (Cicero EM®; Dräger Werke AG, Lübeck, Germany) at normal endtidal PCO2. Fresh gas flow was changed to 70% N2O (n = 38) or air/30% O2 (n = 8) after induction using 100% oxygen. Isoflurane was added to a final end-tidal concentration of 0.89 ± 0.23Vol% in the low flow state. At the end of the procedure oxygenation was returned to 100% oxygen until tracheal extubation.
Breath samples were collected after operation in the recovery room and in the following 19 days. For the measurement of the enantiomers in breath, samples were collected in Tenax filled glass tubes (Gerstel® MPS; Gerstel GmbH, Mülheim/Ruhr Germany) and analyzed via direct thermodesorption gas chromatography mass spectometry as described in detail elsewhere .
Blood samples were drawn in parallel in the recovery room and daily for the following 7 days by venous puncture (K-EDTA Monovette®; Sarstedt GmbH, Nümbrecht, Germany). For determination of the enantiomers in blood samples, blood was transferred into headspace sample vials (2 mL GC Vials®; Klaus Ziemer GmbH, Mannheim, Germany) and stored frozen at −18°C until analysis was performed. A multipurpose-sampler (Gerstel® MPS) was used for the GC-MS analysis (HP 5890/5971 A®; Hewlett Packard, Waldbronn, Germany) as described elsewhere .
Enantiomer fractions are described as percentage of total isoflurane concentration: 100% = %[S(+) enantiomer] + %[R(−) enantiomer]. Only GC-MS measurements with a signal to noise ratio ≥6 were accepted for calculation in this study. The intra-assay coefficient of variation for breath/blood samples was determined as 0.21%/0.7%, respectively. For the determination of the enantiomer ratio in the applied isoflurane racemate, pure not pre-exposed gas or blood samples were enriched for isoflurane enantiomers. The enantiomer ratio of isoflurane racemate was determined in vitro as 50 ± 0.03% in breath and 50 ± 0.1% in blood probes (determined during each 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 to the racemic control was made by Kruskal-Wallis one-way analysis of variance on ranks including Dunn's method for multiple comparisons. In addition for the analysis of the subgroups of patients the Friedman repeated measures analysis of variance on ranks was performed. Further data were analyzed by the Mann-Whitney Rank Sum test.
Forty-six patients (29 females, 17 males) were included in the study. Patient age was 57 ± 15 yr, height 170 ± 8 cm and weight 77 ± 17 kg (mean ± SD). The patients were ASA I-III and underwent different elective operative procedures with a mean duration of 173 ± 61 min. Patients younger than 18 yr, with mental disease, with any isoflurane exposure in the last 3 weeks or who were planned for ambulant procedures were excluded from the study.
Breath samples were collected until 19 days after surgery in up to 46 patients (Table 1). The isoflurane enantiomer in blood samples distribution was analyzed after recovery and in the following seven post-operative days in up to 39 patients (Table 2). Since some patients refused further blood drawing, or sample collection or were unavailable, the numbers of patients vary at the different time points (Table 1 and 2). Further the isoflurane concentration of some blood samples dropped below the detection limit toward day 7.
The amount of S(+) enrichment in breath samples was significantly different to the racemate at all time points investigated (P < 0.001). In the early postoperative phase (recovery room to 4th post-operative day), especially at day 4 (50.49%) S(+) enantiomers were significantly enhanced compared to later time points (P < 0.05) (Figs 1 and 4). Thereafter the ratio of S(+)/R(−) shifted towards the R(−) enantiomers during the remaining time interval investigated (Figs. 1 and 3). Also in blood samples, an enrichment of S(+) occurred; however, it was much more pronounced (compare Tables 1 and 2; Figs 3-4). After extubation a shift from the applied isoflurane racemate (50.04% S(+) and 49.96% R(−)) towards an increase of S(+) enantiomer occurred in blood samples (Fig. 2, Table 2). Levels of enantiomer enrichment were significantly elevated (P < 0.05) compared to the isoflurane racemate blood control samples between days 1 and 5 after surgery (Kruskal-Wallis one-way analysis of variance on Ranks; Dunn's method for multiple comparison). Between days 2 and 4 the enhancement of the S(+) enantiomers was also significantly higher than in the recovery room (day 0) (P < 0.002). Based on the median, the most prominent S(+) enantiomer enrichment (51.43%) was detected 3 days after surgery (P < 0.001) (Mann-Whitney Rank Sum Test) (Figs. 2 and 3; Table 2).
Post hoc, patients were categorized in subgroups according to their body mass index (BMI), application of NO2 and gender. None of these factors had any significant influence on the amount of S(+) isoflurane enantiomers in breath samples (not shown).
Fluorinated inhalation agents, such as halothane, enflurane, and isoflurane, are chiral compounds. There is evidence that the action mode and distribution of the two isoflurane enantiomers are different. S(+) isoflurane enantiomer was more potent in augmenting the flunitrazepam binding to the γ-aminobutyric acid type A (GABAA) receptor, an inhibitory receptor-channel complex . It increased the potency and efficacy of GABA and increased the GABA gated flux significantly more than the R(−) enantiomer . This feature may be responsible for the higher potency of S(+) enantiomer in mice . Further the S(+) enantiomer of isoflurane was about twice as effective on potassium channels, which silences the cell by hyperpolarization compared to R(−) enantiomer. Summarizing these data, it appears that S(+) is more potent maybe because of special binding properties.
In the present study we were able to confirm our previous data, in regard to dominant presence of S(+) enantiomers in blood samples 3 days after surgery (Fig. 2). Since isoflurane is eliminated mostly by lung ventilation, we compared in this study the enantiomer ratio in blood samples to ratios in breath samples by a new detection method. Due to its high sensitivity, it could be determined during a longer postoperative period . In the early postoperative phase (recovery room to day 4 after surgery), the enantiomer ratios in blood and breath processed in parallel, although the part of S(+) enantiomers was higher in blood (Figs. 3 and 4). After day 4 the ratio shifted in breath samples toward the R(−) enantiomers, whereas in blood samples S(+) enantiomers were still over 50%. The shift towards R(−) in blood could be expected after day 8, although at these time points most samples could not be evaluated, since the concentrations were under detection limit (Fig. 3).
The low blood solubility of isoflurane racemate (blood/gas partition coefficient: 1.4) allows a fast adjustment of the alveolar concentration to inspired concentration . Whereas there are several mathematical models for the uptake of volatile anaesthetics [17-20], the elimination of volatile anaesthetics has not been investigated in detail. Persisting isoflurane concentrations in breath or blood samples after such a long period of time after operation has not been described previously. However, Yasuda and colleagues described different transit times and a lower body clearance of isoflurane up to 5 days after exposure in comparison to other anaesthetics . The elimination of a gas largely depends on the solubility of the anaesthetic and on the duration of anaesthesia [22,23]. On recovery the tissue partial pressures are variable. In the vessel-rich tissue equilibrium to alveolar anaesthetic partial pressure will be achieved, whereas in muscle and fat tissue the equilibrium is achieved only after longer anaesthesia. In our study anaesthesia was maintained on the average 173 min (range, 90-450 min). Therefore, we can assume that at least in the vessel-rich tissue an equilibrium was approached but it is likely that the muscle group and/or fat tissue is not yet equilibrated to alveolar partial pressure in each single patient. In this case, these tissue compartments will take up isoflurane from blood as long as the partial pressure of isoflurane in arterial blood is above that in the tissue. In respect to our data it may be speculated that S(+) enantiomers may be present mainly in tissue with fast equilibrium (e.g. vessel-rich group) whereas R(−) enantiomers may be stored preferentially in tissues with slow equilibrium (e.g. fat tissue). As a result, S(+) enantiomers will be released faster from its compartment than the R(−) enantiomers. As soon as the R(−) enantiomers in blood will be below a certain concentration (day 3) the tissue will release R(−) enantiomers into alveoli via the blood stream (Fig. 3). The measurements of the individual enantiomer concentrations would give further information but so far the necessary analytical method has not been established. In addition, no partition coefficient for the isoflurane enantiomers has yet been described.
Xu and colleagues showed that S(+) enantiomers bind with different association and dissociation rates compared to R(−) enantiomers. However, no stereoselective difference in the fraction of isoflurane enantiomers bound to bovine serum albumin, or in their dissociation coefficient, was found in their study . The same group described in a recent study the immobilization of specific bound isoflurane molecules at nicotinic acetylcholine receptors . Although stereoselectivity was not evaluated in this study, stabilization itself seem to be an important mechanism of anaesthetic-induced function and may be partly involved in the dynamics of isoflurane enantiomers .
Conclusively different binding kinetics to receptors [24,27] or distribution or redistribution in certain tissues  seems to be responsible, somehow, for the kinetics of isoflurane enantiomers detected in our study.
Another aspect could be different metabolism of S(+) and R(−) enantiomers. Metabolism of isoflurane involves hepatic, cytochrome P450-mediated oxidation of the α-carbon atom of isoflurane . In enflurane, the metabolic enantiometric selectivity of human liver P450 2E1 for R(−) enantiomer has been described . Therefore, this effect may also contribute to the different kinetics of isoflurane enantiomers demonstrated in this study, although the metabolic rate of isoflurane is very low and would not explain completely our observations .
In conclusion, we have shown that after the application of racemic isoflurane during surgery, isoflurane appears predominantly as S(+) enantiomer during the early postoperative phase in breath samples and in blood. At later time points this ratio is reversed in breath samples towards the R(−) enantiomer while S(+) enrichment in blood is declining. These data suggest that the two isoflurane enantiomers have different tissue distribution and binding properties in human beings.
1. Calvey TN. Chirality and the mode of action of anaesthetics. Eur J Anaesthesiol
2. Hall AC, Lieb WR, Franks NP. Stereoselective and non-stereoselective actions of isoflurane on the GABAA
receptor. Br J Pharmacol
3. Oz M, Tchugunova Y, Dinc M, Dunn SM. Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols. Arch Biochem Biophys
4. Moody EJ, Harris BD, Skolnick P. Stereospecific actions of the inhalation anesthetic isoflurane at the GABAA
receptor complex. Brain Res
5. Franks NP, Lieb WR. Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels. Science
6. Harris BD, Moody EJ, Basile AS, Skolnick P. Volatile anesthetics bidirectionally and stereospecifically modulate ligand binding to GABA receptors. Eur J Pharmacol
7. Jones MV, Harrison NL. Effects of volatile anesthetics on the kinetics of inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurophysiol
8. Quinlan JJ, Firestone S, Firestone LL. Isoflurane's enhancement of chloride flux through rat brain gamma-aminobutyric acid type A receptors is stereoselective. Anesthesiology
9. Lysko GS, Robinson JL, Casto R, Ferrone RA. The stereospecific effects of isoflurane isomers in vivo. Eur J Pharmacol
10. Dickinson R, White I, Lieb WR, Franks NP. Stereoselective loss of righting reflex in rats by isoflurane. Anesthesiology
11. Eger EI, Koblin DD, Laster MJ, et al.
Minimum alveolar anesthetic concentration values for the enantiomers of isoflurane differ minimally. Anesth Analg
12. Schmidt R, Wahl HG, Haberle H, Dieterich HJ, Schurig V. Headspace gas chromatography-mass spectrometry analysis of isoflurane enantiomers in blood samples after anesthesia with the racemic mixture. Chirality
13. Haeberle HA, Wahl HG, Jakubetz H, et al.
Accumulation of S
(+) enantiomer in human beings after general anaesthesia with isoflurane racemate. Eur J Anaesthesiol
14. Eger II EI. The pharmacology of isoflurane. Br J Anaesth
1984; 56 (Suppl. 1):
15. Wahl, HG, Haberle H, Schmidt R, Dieterich HJ, Liebich HM. Analysis of Isoflurane Enantiomers in Human Breath Thermodesorption Gas Chromatography Mass Spectrometry.
23rd International Symposium on Capillary Chromatography 2000.
16. Harris B, Moody E, Skolnick P. Isoflurane anesthesia is stereoselective. Eur J Pharmacol
17. Lerou JG, Dirksen R, Beneken Kolmer HH, Booij LH. A system model for closed-circuit inhalation anesthesia. I. Computer study. Anesthesiology
18. Lerou JG, Booij LH. Model-based administration of inhalation anaesthesia. 2. Exploring the system model. Br J Anaesth
19. Lerou JG, Booij LH. Model-based administration of inhalation anaesthesia. 3. Validating the system model. Br J Anaesth
20. Lerou JG, Verheijen R, Booij LH. Model-based administration of inhalation anaesthesia. 4. Applying the system model. Br J Anaesth
21. Yasuda N, Lockhart SH, Eger II EI, et al.
Kinetics of desflurane, isoflurane, and halothane in humans. Anesthesiology
22. Eger II EI, Johnson BH. Rates of awakening from anesthesia with I-653, halothane, isoflurane and sevoflurane: a test of the effect of anesthetic concentration and duration in rats. Anesth Analg
23. Stoelting RK, Eger II EI. The effects of ventilation and anesthetic solubility on recovery from anesthesia: an in vivo
and analog analysis before and after equilibrium. Anesthesiology
24. Xu Y, Tang P, Firestone L, Zhang TT. 19F nuclear magnetic resonance investigation of stereoselective binding of isoflurane to bovine serum albumin. Biophys J
25. Xu Y, Seto T, Tang P, Firestone L. NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors. Biophys J
26. Eckenhoff RG. Do specific or nonspecific interactions with proteins underlie inhalational anesthetic action? Mol Pharmacol
27. Pohorecki R, Howard BJ, Matsushita M, Stemmer PM, Becker GL, Landers DF. Isoflurane isomers differ in preservation of ATP in anoxic rat hepatocytes. J Pharmacol Exp Ther
28. Bradshaw JJ, Ivanetich KM. Isoflurane: a comparison of its metabolism by human and rat hepatic cytochrome P-450. Anesth Analg
29. Garton KJ, Yuen P, Meinwald J, Thummel KE, Kharasch ED. Stereoselective metabolism of enflurane by human liver cytochrome P450 2E1. Drug Metab Dispos