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ANESTHETIC PHARMACOLOGY

Chirality in Anesthesia I: Minimum Alveolar Concentration of Secondary Alcohol Enantiomers

From the Department of Anesthesia and Perioperative Care, University of California, San Francisco, California.

Address correspondence to James M. Sonner, MD, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to sonnerj{at}anesthesia.ucsf.edu.

	Abstract

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
Most studies of chirality in inhaled anesthetic action have used the enantiomers of isoflurane. These enantiomers are expensive and scarce, which limits studies, such as the preliminary identification of molecular targets of anesthetic action, that can be performed with these isomers. We hypothesized that secondary alcohols (i.e., compounds having a -CH₂-CHOH-CH₃ group) that are experimental anesthetics would show enantioselectivity. To test this hypothesis, we determined the minimum alveolar anesthetic concentration (MAC) of the enantiomers of the homologous series of 2-alcohols from 2-butanol to 2-heptanol in rats. Because these alcohols are partially metabolized to 2-ketones during the course of study (i.e., having a -CH₂-CO-CH₃ group), we independently measured the MAC of the 2-ketones. Assuming additivity of MAC of the ketones with the alcohols, we corrected for the anesthetic effect of the ketones in rats to determine the MAC of the alcohols. We found that the 2-butanol and 2-pentanol isomers were enantioselective. S-(+)-2-butanol had a MAC that was 17% larger than for the R-(-)-enantiomer, whereas S-(+)-2-pentanol had a MAC that was 38% larger than the R-(-)- enantiomer. No stereoselectivity was observed for 2-hexanol and 2-heptanol. These findings may permit studies of chirality in anesthesia, particularly in in vitro systems where metabolism does not occur, using inexpensive volatile compounds.

	Introduction

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
Most studies of chirality in inhaled anesthetic action have used the enantiomers of isoflurane, which was first prepared in enantiomerically pure form in the early 1990s by an asymmetric synthesis (1). Using these enantiomers in rats, Lysko et al. (2) found that isoflurane minimum alveolar anesthetic concentration (MAC) for the R enantiomer was 53% larger than (1.53 times larger) for the S isomer. A subsequent study in rats found a 17% larger MAC for the R isomer, which was not quite significantly more than the MAC of the S isomer (P = 0.06) (3). In rats, the loss of righting reflex differed between enantiomers by 40% ± 8% (4). These results are potentially important to studies of anesthetic mechanisms because enantioselectivity observed in behaviors that those molecules mediate may reflect the enantioselectivity on molecular targets of anesthetic action, such as specific receptors (5). For example, enantioselectivity of isoflurane on –aminobutyric acid (GABA)_A receptors (5–7) supports these receptors as targets of anesthetic action. In addition, enantiomers of isoflurane have been used to address the fundamental question of whether isoflurane acts by a protein or lipid mechanism (8).

The use of the isomers of isoflurane has a practical disadvantage however, in that they are not commercially available and are costly to produce privately, even in small quantities. As a result, the number and type of studies that can be performed with these compounds is limited. To expand the range of studies using chirality that can be performed, in particular, as a test of relevance for anesthetic targets, we identified experimental volatile compounds that are enantioselective with respect to MAC and commercially available.

Our initial list of compounds included substances expected to be anesthetic and commercially available, including: 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 2-chlorobutane, 2-methyl-1-butanol, 3-methyl-2-butanol, 4-methyl-2-pentanol, 1-phenylethanol, 1-phenyl-1-propanol, 2,2,2,-trifluoro-1-phenylethanol, 2-(hydroxymethyl)oxirane (Glycidol), 1,3-butanediol, and 1,2,4-butanetriol. Ultimately, we settled on the homologous series of 2-alcohols from 2-butanol to 2-heptanol because this would allow us to make comparisons among structurally related anesthetics.

The series of 2-alcohols were previously studied in tadpoles using the loss-of-righting reflex (9). Stereoselectivity was not observed for enantiomers from 2-butanol to 2-octanol. However, isoflurane, which has stereoselective effects on righting reflex in rats, was also reported to lack stereoselectivity in tadpoles, again using the righting reflex (see Reference 8 and citations therein). We conjectured that stereoselectivity for the 2-alcohol series might be observed on MAC in rats and, accordingly, examined a series from 2-butanol through 2-heptanol. Additionally, and in accordance with Pfeiffer's rule, we predicted that stereoselectivity would depend on the potency of these compounds that, in turn, would correlate with chain length (10). We also measured the potencies of the corresponding series of ketones. This was essential to the determination of the potencies of the alcohols because a portion of each alcohol was metabolized to the ketone and had to be accounted for in the estimation of the alcohol MAC.

	METHODS

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
With approval of the Institutional Animal Care and Use Committee of the University of California, San Francisco, we studied 2-alcohol and 2-ketone MAC in 398 male 8- to 10-wk-old [Crl:CD(SD)BR] Sprague-Dawley rats, weighing 275–325 g, obtained from Charles River Laboratories (Hollister, CA). Each rat had access to rat chow and tap water before study.

R and S enantiomers of 2-butanol (99% purity for both isomers), 2-pentanol (98% purity for both isomers), 2-hexanol (99% purity for both isomers), 2-heptanol (99% purity for the S isomer; 95% purity for the R isomer), 2-butanone (99% purity for both isomers), 2-pentanone (99% purity for both isomers), 2-hexanone (98% purity for both isomers), and 2-heptanone (99% purity for both isomers) were purchased from Sigma-Aldrich (St. Louis, MO). The racemate was made as a 1:1 mixture by weight of the component enantiomers. All alcohols were administered by i.p. injection. 2-butanol was delivered as a 10% solution in saline, whereas 2-pentanol, hexanol, and heptanol were dissolved in olive oil to a concentration of 5%, 2%, and 5%, respectively. 2-butanol was dosed at 0.8–1.4 g/kg, 2-pentanol at 0.7–0.85 g/kg, 2-hexanol at 0.6–1.1 g/kg, and 2-heptanol at 0.45–0.75 g/kg. 2-butanone was dissolved in saline at a concentration of 10%, whereas the other ketones were dissolved in olive oil (10% for the pentanone and hexanone and 5% for the heptanone). The dose range for i.p. injections were butanone 1–2 g/kg, pentanone 1–1.5 g/kg, 2-hexanone 0.6–1 g/kg, and 2-heptanone 0.5–1 g/kg.

A preliminary dose-ranging study was performed to determine the approximate injected dose of alcohol or ketone that produced immobility 40 min after the injection. No blood samples were taken, and these results are not reported. For the determination of MAC, we gave the approximate dose indicated by the dose-ranging study to the first several rats. Subsequent doses, larger and smaller than this dose, were given as indicated by the responses of the rats to the initial dose (e.g., we gave more alcohol or ketone if most of the rats responded). This process continued until we obtained a crossover range of arterial alcohol or ketone partial pressures that permitted both movement and nonmovement. Less than this range, all rats moved, and more than this range, no rats moved. This process might require as few as 12 rats or as many as 40 rats. Each rat supplied only one data point (i.e., an alcohol concentration and whether the rat did or did not move.)

Each alcohol and each ketone was given in 2 equal doses injected 5 min apart. Immediately after the injection, each rat was placed in an individual clear plastic cylinder to which 1 L/min of oxygen was delivered. Rectal temperature was monitored and maintained between 37°C and 39°C. Forty minutes after the first injection, the tail of each rat was clamped with an alligator clip that was moved back and forth once per second for 1 min or until the animal moved its head or paw, whichever happened first. The response was recorded, and 5–10 mL of aortic blood was drawn into a heparinized plastic syringe. This blood was then transferred into a glass syringe whose barrel was lubricated with a small amount of glycerin to prevent the barrel from sticking. Twenty to 25 mL of air was added to the blood sample. The contents of the syringe were then equilibrated at 37°C for 1 h.

The gas in equilibrium with the blood in the syringe was then injected through a heated injection port into the gas sampling loop of a gas chromatograph equipped with a carbowax column and a flame ionization detector. The column length was 4.6 m for the pentanol and heptanal and the ketones and 9.2 m for the butanol and hexanol. The column width was 0.22 cm. The carrier gas was nitrogen. The detector received 36-45 mL/min of hydrogen and 200–300 mL/min of air.

Alcohol and ketone concentrations were determined by comparison with primary standards made each day. An aliquot (known weight) of alcohol/ketone was aspirated into a flask of known volume. Using the gas laws, the weight was converted to moles and then to volume. Thus, we obtained a known volume of gaseous alcohol/ketone in a known total volume (i.e., this provided a known concentration). A sample of this was injected into the gas chromatograph. The results are accurate (at least consistent) to 1%–2% of the target value.

Ketone MAC was calculated using logistic regression (11). A different method was used to calculate the MAC of the secondary alcohols because blood samples from rats showed two peaks by gas chromatography. This method is described in Appendix A.

Differences in MAC between the R and S isomers were determined by a Student's t-test. P < 0.05 was taken as the significance threshold.

	RESULTS

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
The data for the 2-alcohols are tabulated in Table 1 and for the 2-ketones in Table 2. The alcohol MAC data are presented graphically in Figure 1. Note that the results for the ketones only were required and used to correct the MAC values for the alcohols. For 2-butanol, the S isomer had a MAC 17% larger than the R isomer (P = 0.021). For 2-pentanol, the S isomer had a MAC 38% larger than the R isomer (P = 0.025). No enantioselectivity was observed for 2-hexanol and 2-heptanol.

View larger version (23K): [in this window] [in a new window]   Figure 1. Minimum alveolar anesthetic concentration (MAC) for the enantiomers and racemic mixture of the 2-alcohols decreases with aliphatic chain length. The R isomer is more potent than the S isomer for 2-butanol and 2-pentanol, but there is no enantioselectivity for the isomers of 2-hexanol or 2-heptanol.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
Enantiomers are compounds that are mirror images of each other but are not superimposable. They have identical physical properties, such as melting points, boiling points, densities, refractive indices, and solution properties in achiral media (e.g., solubility and spectroscopic properties), with the exception of their capacity to rotate plane polarized light, which enantiomers do equally, but in opposite directions (12). Chirality is important in biology because most biologically active molecules, such as drugs and neurotransmitters, are chiral as are their biological targets, such as enzymes and ion channels. Consequently, there is often stereoselectivity for one enantiomer over another by virtue of that enantiomer's complementarity with its biological target. Because of the ubiquity of chirality in biological systems, biochemical processes in general are stereoselective (13). For many drugs, it is an empirical observation that stereoselectivity increases with increasing drug potency, a relationship known as Pfeiffer's rule (10).

We found stereoselective differences for two of the four secondary alcohols studied. For 2-butanol, a 17% difference between the R and S isomers was found, whereas 2-pentanol showed a 38% difference between the isomers. In both cases, the R isomer was more potent than the S isomer. For 2-pentanol, the quantitative differences in enantioselectivity are indistinguishable from those reported for the enantiomers of isoflurane on righting reflex, lying between the two reported values for the stereoselectivity of isoflurane on MAC; however, our results are opposite in sign to the results found by Dickinson et al. (4), who found the S isomer more potent than the R isomer for isoflurane. 2-butanol showed somewhat less, but nonetheless significant, enantioselectivity with respect to MAC. We thus succeeded in our effort to find inexpensive, commercially available, and enantioselective experimental anesthetics.

The 2-alcohols were oxidized to 2-ketones, presumably by alcohol dehydrogenase (14). These ketones had anesthetic effects themselves. To determine enantioselective effects of the secondary alcohols, we had to correct for the anesthetic effect of the 2-ketone metabolite. We did so by assuming additivity of the ketone and alcohol. This assumption is justified by observations that MAC values for different anesthetics, including alcohols, are additive (15). The uncorrected MAC values determined by an analysis of the alcohol concentration in the blood and the ketone MAC values are shown in Tables 1 and 2. For uncorrected values, stereoselectivity of the 2-alcohols increases with potency (i.e., aliphatic chain length). This trend reflects the stereoselective oxidation of the enantiomers by alcohol dehydrogenase (14) to the less potent 2-ketones.

For corrected values, our results did not follow Pfeiffer's rule. MAC differences were observed between isomers of the two less potent alcohols 2-butanol and 2-pentanol and not for the two more potent alcohols 2-hexanol and 2-heptanol. However, these results are consistent with previous observations demonstrating stereoselective inhibition by 2-butanol on nicotinic acetylcholine receptors but not by more potent secondary alcohols (16).

In summary, we identified stereoselective differences in MAC in rats for 2-butanol and 2-pentanol of approximately the same size, as previously reported, for isoflurane in animals. These differences open the door to studies of chirality in anesthesia using these readily available experimental anesthetics.

	Appendix 1

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 
In this study, 2-alcohols were metabolized to 2-ketones, which are anesthetics. To calculate the MAC of the alcohol, we had to correct for the MAC of the ketone (K_MAC). This could be accomplished because two peaks were observed by gas chromatography: one peak corresponding to the alcohol initially injected and the other to the ketone produced by oxidation of the secondary alcohol. However, the variability in ketone concentrations was too small to support an analysis using multinomial logistic regression. We accordingly calculated the MAC of the alcohol assuming that the ketone and alcohol were additive in their effect on MAC:

from which alcohol MAC was given by:

where A_MAC is the MAC of the alcohol, A_{MAC(uncorrected)} is the uncorrected MAC of the alcohol determined using logistic regression based on the concentrations of alcohol in blood determined by gas chromatography but not corrected for the presence of the ketone, K_MAC is the MAC of the ketone determined in a separate experiment, and K_Peak is the concentration determined by gas chromatography of the ketone in blood of animals injected with alcohol. Standard errors for A_MAC were determined using error propagation methods, according to the following equation (17):

	Footnotes

 
Dr. Eger is a paid consultant to Baxter Healthcare Corp

Accepted for publication February 8, 2006.

Supported, in part, by grant NIGMS R01 GM069379

	REFERENCES

Top Abstract Introduction METHODS RESULTS DISCUSSION Appendix 1 REFERENCES

 

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