According to the Meyer-Overton hypothesis, affinity for a lipid-like phase determines anesthetic potency (1,2). Meyer and Gottlieb-Billroth (3) and Meyer and Hemmi (4) stated this as a simple equation: some measure of lipophilicity (e.g., the oil/gas partition coefficient) times some measure of anesthetic potency (e.g., MAC–the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) equals a constant. For conventional anesthetics, this product equals 1.82 ± 0.56 atm (mean ± sd) (5).
Some inhaled compounds have lower MAC values (6,7) than would be predicted by their lipid solubilities (transitional compounds) or have no measurable anesthetic effect (as defined by absence of movement in response to a noxious stimulus, and by absence of a capacity to decrease the requirement for a known anesthetic such as desflurane). The latter compounds have been called “nonimmobilizers” (6,7). A low aqueous affinity (i.e., saline/gas partition coefficients usually < 0.02) distinguishes transitional compounds and nonimmobilizers from conventional anesthetics, and this reflection of a lesser polarity has been given as an explanation for their inability to produce anesthesia (8–11): Their less polar nature suggests that they cannot influence events at interfaces because their weak attraction to water means that they cannot achieve sufficient interfacial concentrations.
In contrast to nonimmobilizers, n-alkanols have a great affinity for water (have large saline/gas partition coefficients and are polar compounds), and alkanols are potent anesthetics. The n-alkanol partial pressures required to cause anesthesia are generally much lower than those of most conventional inhaled anesthetics (12). Finally, and immediately relevant to the present report, the product of n-alkanol MAC times their olive oil/gas partition coefficient is a 10th (0.156 ± 0.072 atm) that noted above for conventional anesthetics. Fluorination of alkanols can modify this relationship. Fluorination of n-alkanols can decrease their hydrophilicity and concurrently decrease their potency (13). These results emphasize the importance of both lipophilicity and hydrophilicity to anesthetic potency.
We examined the effect of substitution of sulfur for oxygen in the -OH moiety of n-alkanols on anesthetic potency. Specifically, we examined the series H(CH2)nSH, comparing the results with the series H(CH2)nOH for compounds having three to six carbon atoms. We hypothesized that sulfur substitution for oxygen would decrease hydrophilicity (i.e., would decrease the saline/gas partition coefficient, a reflection of a decrease in polarity) while sustaining lipophilicity. Further, we hypothesized that these changes would produce products of MAC times olive oil partition coefficients that would approach or exceed those of conventional anesthetics. Demonstration of such findings would be consistent with the hypothesis that the site of action at which inhaled anesthetics produce immobilization has both polar and nonpolar characteristics.
Studies in Rats
With approval of the Committee on Animal Research of the University of California, San Francisco, we studied 87 male specific-pathogen-free, Sprague-Dawley rats weighing 300–450 g obtained from Charles River Laboratories (Hollister, CA). Each animal was caged with up to two additional rats, and all had continuous access to standard rat chow and tap water before study. The rats received n-propanol, n-pentanol, or the corresponding alkanethiols in the series from propane to hexane, inclusive. Data for 1-butanol and 1-hexanol were taken from previous reports from this laboratory (12,14). All compounds were given intraperitoneally. Ethanethiol also was studied in a few rats, but was found to be lethal.
Determination of MAC
All compounds were studied at partial pressures permitting and preventing movement in response to a noxious stimulus. N-propanol (5%–10%) and n-pentanol (2%) were dissolved in saline and injected as one or two boluses. The alkanethiols were dissolved in olive oil (10%–50%), and each of those solutions was injected intraperitoneally as a single bolus.
Two to four rats were prepared for each study. Immediately after the injection of the test compound, we placed each rat in a clear plastic cylinder. We delivered oxygen at approximately a 1 L/min flow through a hole piercing a stopper at one end of the cylinder. The other end was open. We placed a rectal probe to permit monitoring of temperature which was maintained between 36.5°C and 38.5°C by application or infrared light or ice to the outside of the cylinder.
At 40 min after the injection of alkanol or alkanethiol, we stimulated the rat’s tail by applying and moving an alligator clamp for up to 1 min, and observing the rat for movement of an extremity or the head. Forty minutes appears to be the time of peak concentration in the rat brain after the intraperitoneal injection of ethanol (15). If no movement occurred, we made an incision in the abdomen to permit withdrawal of 6–11 mL of blood from the aorta, drawing the blood into a heparinized 50-mL syringe. If movement occurred, we considered this equivalent to movement in response to tail clamp, and we administered desflurane (5%–7%) in 1 L/min of oxygen to achieve anesthesia (lack of movement) and then obtained the blood sample.
To determine the partial pressure of the alkanol in aortic blood, we added 20–34 mL of air to the syringe and equilibrated the contents for 60 min in an incubator, at 37°C. The gas phase was analyzed by using gas chromatography for alkanol (see below) by using primary volumetric standards. Because of the great solubility of alkanols in blood, this gas phase value immediately indicated the alkanol partial pressure in blood.
In preliminary studies, we found that, in contrast to the n-alkanols, the solubility of alkanethiols in blood was too small to assume that the above gas phase value indicated the partial pressure in blood. Accordingly, we altered the determination of the partial pressure in blood as follows. The volume of blood obtained was noted, and an equal volume of air was added. This mixture was equilibrated for >50 min at 37°C in the incubator, and the gas phase concentration (C1) was analyzed by using gas chromatography. All of the gas phase was expelled, a second volume of air equal to the volume of blood was added to the blood, and the mixture was again equilibrated. The gas phase concentration (C2) was then analyzed by using gas chromatography. This process was usually repeated a third and, sometimes, a fourth time. Each set of two to four values was analyzed by using least squares regression and assuming an exponential (first order) decay. The correlation coefficients exceeded 0.96 for >90% of the analyses. We extrapolated back to the original gas phase concentration (Co). This was accepted as the partial pressure (concentration at 1 atm) that the rat had experienced at the time of stimulation. The values for Co and C1 were re-estimated by using the equation derived from the least squares regression. We used these to calculate the blood/gas partition coefficient (λ) for the alkanethiols, using the equation: λ = C1/(C0 − C1).
We applied a logistic regression analysis to the resulting data (16). Each rat supplied two values: the response (movement or no movement) and the associated compound partial pressure. The logistic regression analysis supplied a value for the partial pressure producing absence of movement in 50% of rats (the ED50) and the variance (standard error) about this value. We define this ED50 as MAC.
These compounds are highly soluble in olive oil, making it difficult to measure the concentration in the solvent phase by extraction. Accordingly, we added a small aliquot of alkanol to an aliquot of olive oil to derive a calculated solvent concentration. That is, using a knowledge of the density of each compound, we calculated the concentration of the compound in the olive oil (CS). Approximately (because of the large solubility of these compounds in lipid-like material, the exact amount does not matter) 10 mL of the combination of compound in olive oil was placed in 50-mL syringes, 30 mL of air was added, and the mixture was equilibrated in an incubator at 37°C for 2 h. After this time, the gas phase was analyzed for the concentration of the alkanol (CG). The olive oil/gas partition coefficient was calculated as CS/CG.
The method was applied to the determination of the saline/gas partition coefficients for n-propanol. For n-pentanol, the saline/gas partition coefficient was determined by using a modification of previously described techniques (17). For the alkanethiols, the saline/gas partition coefficient was determined by using the technique described above for the determination of alkanethiol blood/gas partition coefficients.
Vapor Pressure Measurements
Vapor pressures were obtained by allowing liquid compound to equilibrate with a gas phase in an enclosed space. A sample was taken from the gas phase and diluted by a known amount. The resulting concentration was determined by using gas chromatographic analysis, and the undiluted concentration was calculated from the diluted concentration times the dilution factor.
Gas Chromatographic Analysis
We used a Gow-Mac 580 or 750 flame ionization detector gas chromatograph (Gow-Mac Instrument Corp., Bridgewater, NJ). The 4.6-m long, 0.22-cm (internal diameter) column was packed with SF-96. The column temperature was adjusted to give reasonable (3–6 min) retention times. The detector was maintained at temperatures approximately 50°C warmer than the column. The carrier flow was nitrogen at 15 mL/min. The detector received 24 mL/min hydrogen and 240 mL/min air. Primary standards were prepared for each compound and the linearity of the response of the chromatograph determined.
We determined means and standard deviations and performed least squares linear regression. We accepted P < 0.05 as indicative of significance.
Some physical and anesthetic properties of the test compounds are given in Tables 1 and 2, including our previously published data for n-butanol and n-hexanol (12). The alkanethiols had saline/gas partition coefficients that were three orders of magnitude smaller than their n-alkanol counterparts, whereas the corresponding oil/gas partition coefficients did not differ remarkably (Fig. 1;Table 2).
All test compounds produced anesthesia (Table 2;Fig. 2), but the potency of the alkanethiols was far less than their alkanol counterparts (i.e., the MAC of the alkanethiols was more than an order of magnitude greater). One outlier value for the butanethiol determination of MAC was discarded. Because of the marked differences in potency but similarity of solubilities in oil (Fig. 3), the products of the oil/gas partition coefficients and MAC differed greatly (Table 2). For the product of MAC times the olive oil/gas partition coefficient, the alkanethiols obeyed the Meyer-Overton hypothesis, having values approximately equal to those obtained previously for conventional anesthetics. Solubilities of the alkanethiols in blood were not affected by molecular size (Table 2).
Our results indicate that substitution of sulfur for oxygen in n-alkanols decreases potency (i.e., increases MAC) 30-fold in association with an approximately 1000-fold decrease in hydrophilicity (decrease in the saline/gas partition coefficient) and minimal changes in lipophilicity (the olive oil/gas partition coefficient). Note that potency is defined by MAC rather than by a concentration in an aqueous or blood phase. We choose MAC because this concentration immediately reflects a partial pressure. Because we allowed sufficient time to permit equilibration between this partial pressure and the site of anesthetic action, we can assume that the MAC values given in Table 2 and Fig. 3 reflect the partial pressure at that site. The same cannot be said for a concentration in an aqueous or blood phase. A concentration in the aqueous or blood phase bears no constant relationship across anesthetics to the content or partial pressure in the anesthetic site of action (unless one believes the site of anesthetic action is mimicked by saline); aqueous and blood phases serve as conduits to the site of action but do not provide insights into the character of that site.
For nearly 100 years, theories of anesthetic action, including those attempting to explain the action of alkanols, focused on the finding by Meyer and Overton (1,2) that lipophilicity predicted anesthetic potency (e.g., the reciprocal of MAC). Thus, MAC times olive oil/gas partition coefficient equals a constant of approximately 1.82 atm in rats (5). The discovery of volatile or gaseous compounds that had significant lipophilicity but did not produce anesthesia (nonimmobilizers) (6,7,18) called into question the generalizability of the Meyer-Overton observation. The finding that n-alkanols are 10-fold more potent than would be predicted from their lipophilicity similarly impacted on generalizability.
The findings for both nonimmobilizers and alkanols suggested that hydrophilicity also influences potency, and that anesthetics may act at a locus possessing both polar and nonpolar attributes, a suggestion made previously by others (19). That is, anesthesia requires an affinity of anesthetic molecules for both polar and nonpolar phases. Such an interpretation is consistent with the present data. Our results suggest that the -OH moiety augments the production of anesthesia because it provides a higher affinity to the postulated polar site of general anesthetic action and that substitution of the element below oxygen in the periodic table (sulfur) markedly decreases both polarity and anesthetic potency.
For n-alkanethiols, n-alkanols, and n-alkanes from three to six carbon atoms, the addition of each methylene (-CH2-) group increases potency (i.e., decreases MAC) by a factor of approximately 2.6 (Fig. 2). The increase in potency with the addition of each methylene group may be calculated in terms of kcal/mol (20), the resulting values being −0.68 kcal · mole–1 · CH2–1 for the alkanols and −0.58 kcal · mole–1 · CH2–1 for the alkanethiols. The increase is proportional to the increase in lipophilicity (Fig. 1), indicating that this aspect of potency is governed by an attraction to a nonpolar phase, one that appears to be independent of the differences produced by differences among the groups in attraction to a polar phase such as saline. For all of these classes of compounds, an increase in carbon atoms decreases vapor pressure more than it decreases MAC (Tables 1 and 2), indicating that, at some carbon chain length, the vapor pressure would be insufficient to produce anesthesia.
In summary, we found that substitution of sulfur for the oxygen in n-alkanols decreased polarity and potency while sustaining lipophilicity, changes that produced products of MAC times olive oil partition coefficients approximating those of conventional anesthetics. Such findings support the notion that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) results from their greater polarity.
1. Meyer HH. Theorie der alkoholnarkose. Arch Exptl Pathol Pharmakol 1899; 42: 109–18.
2. Overton E. Studien ü ber die narkose, zugleich ein beitrag zur allgemeinen pharmakologie. Jena: Gustav Fischer, 1901: 1–195.
3. Meyer KH, Gottlieb-Billroth H. Theorie der narkose durch inhalationsanästhetika. Z Physiol Chem 1920; 112: 55–79.
4. Meyer HK, Hemmi H. Beiträge zur theorie der narkose. III. Biochem Z 1935; 277: 39–71.
5. Taheri S, Halsey MJ, Liu J, et al. What solvent best represents the site of action of inhaled anesthetics in humans, rats and dogs? Anesth Analg 1991; 72: 627–34.
6. Koblin DD, Chortkoff BS, Laster MJ, et al. Polyhalogenated and perfluorinated compounds that disobey the Meyer-Overton hypothesis. Anesth Analg 1994; 79: 1043–8.
7. Liu J, Laster MJ, Koblin DD, et al. A cut-off in potency exists in the perfluoroalkanes. Anesth Analg 1994; 79: 238–44.
8. Pohorille A, Cieplak P, Wilson MA. Interactions of anesthetics with the membrane-water interface. Chem Phys 1996; 204: 337–45.
9. Eger EI II, Koblin DD, Harris A, et al. Hypothesis: inhaled anesthetics produce immobility and amnesia by different mechanisms at different sites. Anesth Analg 1997; 84: 915–8.
10. Chipot C, Wilson MA, Pohorille A. Interactions of anesthetics with the hexane-water interface. J Phys Chem B 1996; 101: 782–91.
11. Pohorille A, Wilson MA, Cieplak P. Interaction of alcohols and anesthetics with the water-hexane interface: a molecular dynamics study. Prog Colloid Polym Sci 1997; 103: 29–40.
12. Fang ZX, Ionescu P, Chortkoff BS, et al. Anesthetic potencies of n-alkanols: results of additivity studies suggest a mechanism of action similar to that for conventional inhaled anesthetics. Anesth Analg 1997; 84: 1042–8.
13. Eger EI II, Ionescu P, Laster MJ, et al. MAC of fluorinated alkanols in rats: relevance to theories of narcosis. Anesth Analg 1999; 88: 867–76.
14. Fang ZX, Gong D, Ionescu P, et al. Maturation decreases ethanol MAC more than desflurane MAC in rats. Anesth Analg 1997; 85: 852–8.
15. Yoshimoto K, Komura S. Monitoring of ethanol levels in the rat nucleus accumbens by brain microdialysis. Alcohol Alcoholism 1993; 28: 171–4.
16. Waud DR. On biological assays involving quantal responses. J Pharmacol Exper Therap 1972; 183: 577–607.
17. Taheri S, Laster MJ, Liu J, et al. Anesthesia by n-alkanes not consistent with the Meyer-Overton hypothesis: determinations of the solubilities of alkanes in saline and various lipids. Anesth Analg 1993; 77: 7–11.
18. Eger EI II, Liu J, Koblin DD, et al. Molecular properties of the “ideal” inhaled anesthetic: studies of fluorinated methanes, ethanes, propanes and butanes. Anesth Analg 1994; 79: 245–51.
19. Hansch C, Vittoria A, Silipo C, Jow PY. Partition coefficients and the structure-activity relationship of the anesthetic gases. J Med Chem 1975; 18: 546–8.
20. Dickinson R, Franks NP, Lieb WR. Thermodynamics of anesthetic/protein interactions: temperature studies on firefly luciferase. Biophys J 1993; 64: 1264–71.