MAC (the minimum alveolar concentration of anesthetic producing unresponsiveness to noxious stimulation in 50% of subjects) progressively decreases in the series CH2F2, CHF2CHF2, CHF2CF2CHF2, and CHF2CF2CF2CHF2, each step in the series producing an approximately 2.5-fold increase in potency (Fig. 1) (1). MAC for CHF2(CF2)2CHF2 is 0.058 ± 0.016 atm. However, a discontinuity arises with further increase in chain length: the hexane in the series -CHF2(CF2)4CHF2 (1,6-dihydroperfluorohexane)- is a less potent compound (MAC > 0.175 atm) and probably is a nonimmobilizer (2). Similarly, the MAC for CHF2(CF2)3CH2OH is approximately six times smaller than the MAC for CHF2CF2CH2OH, but the MAC for CHF2(CF2)5CH2OH is larger than that for either of the smaller alkanols (3). Furthermore, studies with perfluorinated n-alkanes and alkanols and with monohydroperfluoroalkanes indicate that the −CF3 and −CF2− moieties have minimal or no anesthetic effect in the absence of an added hydrogen or in the absence of an adjacent carbon with an attached hydrogen (1,4). For example, the addition of ―CF3 and ―CF2― to ―CHF2 (to produce CF3CHF2 or CF3CF2CHF2) does not decrease MAC. That is, the ―CHF2 and ―CH2OH moieties supply the anesthetizing capacity of monohydrogenated and dihydrogenated perfluoroalkanes and monohydrogenated perfluoroalkanols. Finally, perfluorination produces compounds that are relatively rigid rods (i.e., the ―CF2― groups allow minimal flexibility), in contrast to the flexibility present in unhalogenated n-alkanes or n-alkanols (5–7).
In summary, dihydrogenated perfluoroalkanes and monohydrogenated perfluoroalkanols increase in potency for each series for chain lengths of up to four or five carbon atoms but decrease with further extensions in length. Such a low cutoff in potency is not seen for unhalogenated alkanes or alkanols, or for other organic compounds. These findings for dihydrogenated perfluoroalkanes and monohydrogenated perfluoroalkanols are consistent with two sites of anesthetic action separated by five carbon atoms (5 Å) that jointly mediate the capacity of inhaled anesthetics to produce immobility (2).
However, another interpretation is possible. Perhaps the cutoff seen above five carbon atoms results from the rigidity of the perfluorinated structures. This rigidity differs from that found for n-alkanes or n-alkanols, which are flexible structures. Perhaps the rigidity prevents larger perfluorinated structures from occupying, and thereby affecting, a pocket in the protein structure mediating the anesthetic effects of these compounds. In contrast, the flexibility of n-alkanes or n-alkanols allows them to conform to that pocket.
Some unhalogenated alkenes form rigid straight rods. When trans, trans-trans, and trans-trans-trans n-alkenes are conjugated (i.e., in the simplest example, they have alternating single and double bonds), they constitute such compounds. The carbon atoms connected by unsaturation lie in a line, and if the carbon atoms attached to them are in the trans position, then all four carbon atoms zigzag across that line. Cis compounds are also more rigid than saturated compounds, but they are curved. Compounds with methylene groups intermediate to carbon atoms that connect to other carbon atoms by unsaturated bonds contribute flexibility. If rigidity prevents the larger perfluorinated structures from occupying, and thereby affecting, a pocket in the protein structure mediating the anesthetic effects of these compounds, then a larger rigid unhalogenated n-alkane should similarly be less potent than smaller rigid unhalogenated n-alkanes—or not anesthetic at all. However, if the presence of the fluorinated (nonanesthetizing) coat is what prevents CHF2CF2CF2CF2CF2CHF2 from producing anesthesia, then 2,4-trans-trans-hexadiene (CH3-CH=CHCH=CHCH3) should produce anesthesia. That is, the fluorine coat produces different intermolecular attractions from that offered by the trans-trans compound. We tested these predictions in the work presented here.
With approval of the Committee on Animal Research of the University of California, San Francisco, we studied 72 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 compounds studied were ethylene (CH2=CH2), acetylene (CH=CH), 2-trans-butene (CH3CH=CH-CH3), 2-cis-butene (CH3CH=CHCH3), trans-piperylene (CH2=CHCH=CHCH3), cis-piperylene (CH2=CH-CH=CHCH3), 1,4-pentadiene (CH2=CHCH2CH=CH3), 2,4-trans-trans-hexadiene (CH3CH=CHCH=CH-CH3), 1-hexyne (CH=CHCH2CH2CH2CH3), 3-hexyne (CH3CH2C=CCH2CH3), 1,5-hexadiene (CH2=CHCH2-CH2CH=CH3), and 1,3,5-trans-trans-trans-heptatriene (CH2=CHCH=CHCH=CHCH3). Most compounds were >99% pure. Several were purchased from Aldrich Chemical Co. (Milwaukee, WI). Acetylene (>99% pure in acetone; the acetone was removed before use by passing the gas through a water trap) was purchased from Praxair, Inc. (Oak Brook, IL). 2,4-Trans-trans-hexadiene (98% pure) and 1,5-hexadiene (97% pure) were purchased from Pfaltz & Bauer, Inc. (Waterbury, CT). 1-Hexyne (>99% pure) was specifically synthesized for this study by Petra Research, Inc. (Alachua, FL). 1,3,5-Trans-trans-trans-heptatriene (99% pure) was purchased from Synquest Labs, Inc. (Alachua, FL).
All compounds were studied to obtain partial pressures permitting and preventing movement in response to a noxious stimulus. The concentration midway between the largest concentration permitting and the smallest concentration preventing movement provided the MAC value for individual rats. However, 1,3,5-trans-trans-trans-heptatriene MAC values were limited in their usefulness because this compound was lethal at concentrations that caused anesthesia. We attempted to determine the MAC of this compound by studying the decrease in isoflurane MAC that a constant concentration of 1,3,5-trans-trans-trans-heptatriene produced. MAC for isoflurane was determined in an initial study on a different day from the day of study with 1,3,5-trans-trans-trans-heptatriene.
For each study, two rats, each enclosed in its individual plastic cylinder, were placed into a 3.4-L clear plastic cylinder (permitting visualization of each rat) having metal ends containing O-rings and clamps to provide a seal. This closed system allowed the administration and sampling of the inhaled drug, the administration of oxygen (typically maintained at 0.6–1.0 atm), removal of carbon dioxide with soda lime, circulation of gases through the closed system with a fan, and monitoring of rectal temperatures (maintained between 36°C and 39°C by application of ice or infrared light). The chamber was flushed with oxygen for a sufficient time to increase the chamber concentration to >98% oxygen as measured by a Rascal monitor (5250 Ohmeda) (Ohmeda, Madison, WI).
Saline/gas partition coefficients were determined with standard techniques (8). These techniques also were applied to the study of the solubility of 1-hexyne and 3-hexyne in blood. Olive oil/gas partition coefficients for gaseous compounds were also determined with these techniques. For some of the larger compounds, we measured blood/gas partition coefficients with an alternative technique. We drew blood from nonstudy rats into heparinized syringes. The volume of blood obtained was noted, and compound vapor was added in an equal volume of air. This mixture was equilibrated for >50 min at 37°C in an incubator, and the gas phase concentration (C1) was analyzed by 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 gas chromatography. This process was repeated a third and, sometimes, a fourth time. Each set of three 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 (C0). The values for C0 and C1 were reestimated by using the equation derived from the least-squares regression analysis. We used these to calculate the blood/gas partition coefficient (λ) with the equation: λ = C1/(C0 − C1).
The solubility in olive oil of compounds larger than four carbons was too great to allow accurate measurement of the partition coefficient by extraction from the solvent phase. Accordingly, we added a small aliquot of each compound to an aliquot of olive oil to derive a calculated solvent concentration. That is, by using a knowledge of the density of each compound (determined separately), we calculated the concentration of the compound in the olive oil (CS). Approximately (because of the large solubility in olive oil, the exact amount does not matter) 15 mL of the combination of compound in olive oil was placed into 50-mL syringes, 25 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. Solubilities were determined in triplicate or quadruplicate.
For most determinations, 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 (inner diameter [ID]) column was packed with stationary phase-96. 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. We analyzed acetylene and ethylene with a thermal conductivity detector. The 3.05-m-long, 0.22-cm (ID) column contained Haysep D 100/200 mesh (Hayes Separations, Bandera, TX). We analyzed 1,4-pendadiene with a flame ionization detector. The 1.83-m-long, 0.22-cm (ID) column contained Poropak Q, 80/100 mesh (Waters, Milford, MA). Primary standards were prepared for each compound, and the linearity of the response of the chromatograph was determined.
We determined mean and sd, performed Student’s t-tests on unpaired data, and performed least-squares linear regression analyses. We accepted P < 0.05 as significant for the t-test analyses.
Some physical and anesthetic properties of the test compounds are given in Tables 1 and 2. The data include our previously published data for the n-alkanes methane, ethane, butane, and hexane (9) and for 1, n-dihydroperfluorinated compounds (1). All test compounds produced anesthesia. However, as noted above, 1,3,5-trans-trans-trans-heptatriene proved to be both anesthetic and (shortly after having found unresponsiveness) lethal in four rats at 0.0093 ± 0.0013 atm (mean ± sd). Smaller concentrations of 1,3,5-trans-trans-trans-heptatriene appeared to produce early stages of anesthesia, with increasing concentrations causing progressive sedation and concurrent decreases in temperature. No twitching was seen. Studies of additivity with isoflurane with this compound at partial pressures one tenth of those that caused death when given alone (0.00088 ± 0.00019 atm) decreased isoflurane MAC by 2.1% ± 8.3% (decrease not significant). In this additivity study, 5 of 12 rats studied died during or soon after exposure. Thus, the results obtained cannot be used with confidence to provide information on the MAC for this compound.
Values for blood/gas partition coefficients (Table 1) suggested that the inspired concentration measured for these anesthetics did not immediately reflect their partial pressures in arterial blood. Thus, the inspired values were corrected by using the work of Liu et al. (10), who demonstrated the correlation between the blood/gas partition coefficient and the ratio of arterial (Pa) to inspired (PI) anesthetic partial pressures. The equation used for this correction is Pa/PI = 0.933 times the natural log increased to (−0.0953 times the blood/gas partition coefficient). Because we did not measure the solubilities for the two- and four-carbon compounds, we assumed that the correction would be the same as that found by Liu et al. (10) for ethane and butane, respectively. The corrected MAC values are given in Table 2.
None of the solubilities of these compounds was remarkable (Table 1). The saline/gas partition coefficients easily exceeded those found with nonimmobilizer or transitional compounds (11). The oil/gas partition coefficients spanned two or three orders of magnitude, predictive of a similar span of potencies (Table 2).
The addition of unsaturated bonds to n-alkanes to produce alkenes or alkynes did not appear to cause discontinuities in potency. The progressive increase in potency seen with n-alkanes with an increase in carbon atoms is also seen with alkenes and alkynes (Fig. 1). These results contrast with those found with 1, n-dihydroperfluoroalkanes, where the MAC for 1,6-dihydroperfluorohexane significantly exceeded that for the butane (also Fig. 1). Although rectilinear n-alkenes containing five and six carbon atoms are more rigid than n-alkanes, the rectilinear n-alkenes are as potent in the provision of anesthesia (Table 2). That is, such rigidity does not prevent these compounds from entering into and thereby affecting the structure that mediates their anesthetic effects. These findings thus are consistent with our interpretation of previous results for dihydrogenated perfluoroalkanes and monohydrogenated perfluoroalkanols (2). We suggest that anesthesia as defined by MAC results from a concurrent action on two sites separated by five carbon atoms (five angstroms).
However, the results of this study did not provide as rigorous a test of our thesis as it might have had we been able to adequately test the anesthetic properties of 1,3,5-trans-trans-trans-heptatriene. This compound would have easily extended beyond the five-angstrom distance that we suggest is crucial to the two sites of anesthetic action. If 1,3,5-trans-trans-trans-heptatriene is an anesthetic, we know its MAC is more than 0.0093 atmospheres, the lethal inspired partial pressure. This partial pressure should be corrected to an arterial partial pressure of 0.0051 atmospheres because of its solubility. Such a partial pressure is consistent with its size (Fig. 1). Were 0.0051 atmospheres actually the anesthetizing partial pressure, the Meyer-Overton constant (the product of some measure of potency, such as MAC, and some measure of lipophilicity, such as the oil/gas partition coefficient) would be 8.6 atmospheres, a high value suggesting that 1,3,5-trans-trans-trans-heptatriene is, at least, a transitional compound (11) (i.e., a compound less potent than would be predicted from its lipophilicity).
None of our results suggests that an increase in rigidity materially decreases anesthetic potency. For a given number of carbon atoms, the more rigid, unsaturated compounds have the same (or more) potency (smaller MAC) as their saturated peers (Table 2). Compounds having triple bonds are more potent than their peers (Table 2, Fig. 1), and the triple bond confers great rigidity. The increased potency may also be seen in terms of the Meyer-Overton constant. The constant (last column, Table 2) tends to be less for unsaturated than for peer-saturated compounds and is least for compounds having triple bonds (Fig. 2). Finally, note that the potency of 3-hexyne is more than the potency of 1-hexyne. Although these two compounds have the same degree of unsaturation, the rectilinear rigidity of 3-hexyne extends over four carbon atoms (Carbons 2, 3, 4, and 5), whereas the rigidity of 1-hexyne extends over only three (Carbons 1, 2, and 3). Again, more rigidity does not diminish potency.
We also can compare the potencies of more rigid structures that are curved (e.g., the cis compounds) with those that are rectilinear (the trans compounds). 2-Cis-butene does not differ in potency from 2-trans-butene (Table 2). Cis-piperylene is more potent than trans-piperylene (P < 0.001 by an unpaired t-test), but 1,5-hexadiene does not differ in potency from the rigid peer 2,4-trans-trans-hexadiene.
Our arguments assume that -CF2- groups produce more rigidity than -CH2- groups and that the imposition of unsaturated bonds produces still more rigidity (12). For saturated hydrocarbons, there is hindered rotation around C-C bonds. For example, butane can assume any of three positions: one “anti” (low energy, elongated zigzag) and two “gauche” (bent left-handed and right-handed positions of equal energy that are slightly higher than that for the “anti” state). Although butane can assume all three conformational states, the probability of each gauche state is a fraction of that of the trans state, the fraction determined by the Boltzmann factor being approximately 0.3 for butane (12). Replacement of hydrogens with fluorines, particularly with perfluorination (i.e., -CF2- for -CH2-) increases the energy gap between gauche and anti states, leading to a marked preference for the latter and thus an increased likelihood of a straightened, stiffer molecule that follows a zigzag line. But some bending (a gauche state) still can occur. Unsaturation locks the molecule into one conformational state and thereby further increases rigidity. Possible exchange of positions (gauche to anti and back) can occur with saturated hydrocarbons, but with unsaturated hydrocarbons there is no exchange of positions of the hydrogen atoms or any atoms that might substitute for them. Thus, 1,2-dichloroethylene is two molecules, one with both chlorines on the same side (cis) and one on opposite sides (trans). Analogously, there is cis- and trans-2-butene, in which the four carbons and two hydrogens lie in a plane, in the cis case bent and in the trans case following a straight course through a zigzag line. The zigzag line that results with trans compounds having double bonds differs from the straight line resulting from triple bonds. The latter produce truly rectilinear molecules for the two carbon atoms and the atoms immediately attached to each carbon. Regardless, both double and the triple bonds produce a near-absolute rigidity because of the inability of the attached atoms to exchange positions.
We conclude that molecular flexibility is not a necessary structural requirement for anesthetic action, at least for compounds having six carbon atoms or fewer. We also conclude that unsaturation, particularly a triple bond, increases anesthetic potency defined either by MAC or the Meyer-Overton constant. Our results add support to our previous data, which led to the hypothesis that anesthesia results from an action on two molecular sites separated by five angstroms.
We appreciate the several helpful comments made by Professor Robert Cantor, Dartmouth University.
1. 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.
2. Eger EI II, Halsey MJ, Harris RA, et al. Hypothesis: volatile anesthetics produce immobility by acting on two sites approximately five carbons apart. Anesth Analg 1999; 88: 1395–400.
3. 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.
4. Liu J, Laster MJ, Koblin DD, et al. A cut-off in potency exists in the perfluoroalkanes. Anesth Analg 1994; 79: 238–44.
5. Bunn CW, Howells ER. Structures of molecules and crystals of fluorocarbons. Nature 1954; 174: 549–51.
6. Smith GD, Jaffe RL, Yoon DY. Conformational characteristics of poly(tetrafluoroethylene) chains based upon ab initio electronic structure calculations on model molecules. Macromolecules 1994; 27: 3166–73.
7. Eaton DF, Smart BE. Are fluorocarbon chains “stiffer” than hydrocarbon chains? Dynamics of end-to-end cyclization in a C8
segment monitored by fluorescence. J Am Chem Soc 1990; 112: 2821–3.
8. 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.
9. Liu J, Laster MJ, Taheri S, et al. Is there a cutoff in potency for the normal alkanes? Anesth Analg 1993; 77: 12–8.
10. Liu J, Laster MJ, Taheri S, et al. Effect of n
-alkane kinetics in rats on potency estimations and the Meyer-Overton hypothesis. Anesth Analg 1994; 79: 1049–55.
11. Koblin DD, Chortkoff BS, Laster MJ, et al. Polyhalogenated and perfluorinated compounds that disobey the Meyer-Overton hypothesis. Anesth Analg 1994; 79: 1043–8.
© 2001 International Anesthesia Research Society
12. Flory PJ. Statistical mechanics of chain molecules. New York: Wiley Interscience, 1969.